Adeno-associated virus (aav) clades, sequences, vectors containing same, and uses therefor

ABSTRACT

Sequences of novel adeno-associated virus capsids and vectors and host cells containing these sequences are provided. Also described are methods of using such host cells and vectors in production of rAAV particles. AAV-mediated delivery of therapeutic and immunogenic genes using the vectors of the invention is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/573,600, filed Mar. 24, 2006, which is a national stage applicationunder 35 USC §371 of PCT/US04/028817, filed Sep. 30, 2004, which claimsthe benefit under 35 USC §119(e) of the priority of U.S. PatentApplication No. 60/508,226, filed Sep. 30, 2003 and U.S. PatentApplication No. 60/566,546, filed Apr. 29, 2004. Each of theseapplications is hereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This application contains work supported by grants NIDDK P30 DK47757 andNHLBI P01 HL59407 from the National Institutes of Health (NIH). The USgovernment may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV), a member of the Parvovirus family, is asmall nonenveloped, icosahedral virus with single-stranded linear DNAgenomes of 4.7 kilobases (kb) to 6 kb. AAV is assigned to the genus,Dependovirus, because the virus was discovered as a contaminant inpurified adenovirus stocks. AAV's life cycle includes a latent phase atwhich AAV genomes, after infection, are site specifically integratedinto host chromosomes and an infectious phase in which, following eitheradenovirus or herpes simplex virus infection, the integrated genomes aresubsequently rescued, replicated, and packaged into infectious viruses.The properties of non-pathogenicity, broad host range of infectivity,including non-dividing cells, and potential site-specific chromosomalintegration make AAV an attractive tool for gene transfer.

Recent studies suggest that AAV vectors may be the preferred vehicle forgene delivery. To date, there have been several differentwell-characterized AAVs isolated from human or non-human primates (NHP).

It has been found that AAVs of different serotypes exhibit differenttransfection efficiencies, and also exhibit tropism for different cellsor tissues. However, the relationship between these different serotypeshas not previously been explored.

What is desirable are AAV-based constructs for delivery of heterologousmolecules.

SUMMARY OF THE INVENTION

The present invention provides “superfamilies” or “clades” of AAV ofphylogenetically related sequences. These AAV clades provide a source ofAAV sequences useful for targeting and/or delivering molecules todesired target cells or tissues.

In one aspect, the invention provides an AAV clade having at least threeAAV members which are phylogenetically related as determined using aNeighbor-Joining heuristic by a bootstrap value of at least 75% (basedon at least 1000 replicates) and a Poisson correction distancemeasurement of no more than 0.05, based on alignment of the AAV vp1amino acid sequence. Suitably, the AAV clade is composed of AAVsequences useful in generating vectors.

The present invention further provides a human AAV serotype previouslyunknown, designated herein as clone 28.4/hu.14, or alternatively, AAVserotype 9. Thus, in another aspect, the invention provides an AAV ofserotype 9 composed of AAV capsid which is serologically related to acapsid of the sequence of amino acids 1 to 736 of SEQ ID NO: 123 andserologically distinct from a capsid protein of any of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7 or AAV8.

Vectors constructed with capsid of this huAAV9 have exhibited genetransfer efficacies similar to AAV8 in liver, superior to AAV1 in muscleand 200 fold higher than AAV 5 in lung. Further, this novel human AAVserotype shares less than 85% sequence identity to previously describedAAV1 through AAV8 and is not cross-neutralized by any of these AAVs.

The present invention also provides other novel AAV sequences,compositions containing these sequences, and uses therefor.Advantageously, these compositions are particularly well suited for usein compositions requiring re-administration of AAV vectors fortherapeutic or prophylactic purposes.

These and other aspects of the invention will be readily apparent fromthe following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a tree showing the phylogenic relationship constructed usingthe Neighbor-Joining heuristic with Poisson correction distancemeasurement. The relationship was determined based on the isolated AAVvp1 capsid protein, with the isolated AAV grouped in clades. Groups ofindividual capsid clones are classified in clades based on their commonancestry. Clade nomenclature goes from A through F; subtypes arerepresented by the clade letter followed by a number.

FIGS. 2A-2AE are an alignment of the amino acid sequences of AAV vp1capsid proteins of the invention, with the numbering of the individualsequences reported, and previously published AAV1 [SEQ ID NO: 219]; AAV2[SEQ ID NO: 221]; AAV3-3 [SEQ ID NO: 217]; AAV4-4 [SEQ ID NO: 218]; AAV5[SEQ ID NO: 216]; AAV6 [SEQ ID NO: 220]; AAV7 [SEQ ID NO: 222]; AAV8[SEQ ID NO: 223], and; rh. 25/42-15; 29.3/bb.1; cy.2; 29.5/bb.2; rh.32,rh.33, rh.34, rh.10; rh.24; rh14, rh.16, rh.17, rh.12, rh.18, rh.21(formerly termed 41.10); rh.25 (formerly termed 41.15); rh2; rh.31;cy.3; cy.5; rh.13; cy.4; cy.6; rh.22; rh.19; rh.35; rh.37; rh.36; rh.23;rh.8; and ch.5 [US Published Patent Application No. 2003/0138772 A1(Jul. 24, 2003)]. The sequences of the invention include hu.14/AAV9 [SEQID NO:123]; hu.17 [SEQ ID NO: 83], hu. 6 [SEQ ID NO: 84], hu.42 [SEQ IDNO: 85], rh.38 [SEQ ID NO: 86], hu.40 [SEQ ID NO: 87], hu.37 [SEQ ID NO:88 ], rh.40 [SEQ ID NO: 92], rh.52 [SEQ ID NO: 96]; rh.53 [SEQ ID NO:97]; rh.49 [SEQ ID NO: 103]; rh.51 [SEQ ID NO: 104]; rh.57 [SEQ ID NO:105]; rh.58 [SEQ ID NO: 106], rh.61 [SEQ ID NO: 107]; rh.50 [SEQ ID NO:108]; rh.43 [SEQ ID NO: 163]; rh.62 [SEQ ID NO: 114]; rh.48 [SEQ ID NO:115]; 4-9/rh.54 (SEQ ID No: 116); and 4-19/rh.55 (SEQ ID Nos: 117);hu.31 [SEQ ID NO:121]; hu.32 [SEQ ID NO:122]; hu.34 [SEQ ID NO: 125];hu.45 [SEQ ID NO: 127]; hu.47 [SEQ ID NO: 128]; hu.13 [SEQ ID NO:129];hu.28 [SEQ ID NO:130]; hu.29 [SEQ ID NO:132]; hu.19 [SEQ ID NO: 133];hu.20 [SEQ ID NO: 134]; hu.21 [SEQ ID NO:135]; hu.23.2 [SEQ ID NO:137];hu.22 [SEQ ID NO: 138]; hu.27 [SEQ ID NO: 140]; hu.4 [SEQ ID NO: 141];hu.2 [SEQ ID NO: 143]; hu.1 [SEQ ID NO: 144]; hu.3 [SEQ ID NO: 145];hu.25 [SEQ ID NO: 146]; hu.15 [SEQ ID NO: 147]; hu.16 [SEQ ID NO: 148];hu.18 [SEQ ID NO: 149]; hu.7 [SEQ ID NO: 150]; hu.11 [SEQ ID NO: 153];hu.9 [SEQ ID NO: 155]; hu.10 [SEQ ID NO: 156]; hu.48 [SEQ ID NO: 157];hu.44 [SEQ ID NO: 158]; hu.46 [SEQ ID NO: 159]; hu.43 [SEQ ID NO: 160];hu.35 [SEQ ID NO: 164]; hu.24 [SEQ ID NO: 136]; rh.64 [SEQ ID NO: 99];hu.41 [SEQ ID NO: 91]; hu.39 [SEQ ID NO: 102]; hu.67 [SEQ ID NO: 198];hu.66 [SEQ ID NO: 197]; hu.51 [SEQ ID NO: 190]; hu.52 [SEQ ID NO: 191];hu.49 [SEQ ID NO: 189]; hu.56 [SEQ ID NO: 192]; hu.57 [SEQ ID NO: 193];hu.58 [SEQ ID NO: 194]; hu.63 [SEQ ID NO: 195]; hu.64 [SEQ ID NO: 196];hu.60 [SEQ ID NO: 184]; hu.61 [SEQ ID NO: 185]; hu.53 [SEQ ID NO: 186];hu.55 [SEQ ID NO: 187]; hu.54 [SEQ ID NO: 188]; hu.6 [SEQ ID NO: 84];and rh.56 [SEQ ID NO: 152]. These capsid sequences are also reproducedin the Sequence Listing, which is incorporated by reference herein.

FIGS. 3A-3CN are an alignment of the nucleic acid sequences of AAV vp1capsid proteins of the invention, with the numbering of the individualsequences reported, and previously published AAV5 (SEQ ID NO: 199);AAV3-3 (SEQ ID NO: 200); AAV4-4 (SEQ ID NO: 201); AAV1 (SEQ ID NO: 202);AAV6 (SEQ ID NO: 203); AAV2 (SEQ ID NO: 211); AAV7 (SEQ ID NO: 213) andAAV8 (SEQ ID NO: 214); rh. 25/42-15; 29.3/bb.1; cy.2; 29.5/bb.2; rh.32,rh.33, rh.34, rh.10; rh.24; rh14, rh.16, rh.17, rh.12, rh.18, rh.21(formerly termed 41.10); rh.25 (formerly termed 41.15; GenBank accessionAY530557); rh2; rh.31; cy.3; cy.5; rh.13; cy.4; cy.6; rh.22; rh.19;rh.35; rh.37; rh.36; rh.23; rh.8; and ch.5 [US Published PatentApplication No. 2003/0138772 A1 (Jul. 24, 2003)]. The nucleic acidsequences of the invention include, hu.14/AAV9 (SEQ ID No: 3);LG-4/rh.38 (SEQ ID No: 7); LG-10/rh.40 (SEQ ID No: 14); N721-8/rh.43(SEQ ID No: 43); 1-8/rh.49 (SEQ ID NO: 25); 2-4/rh.50 (SEQ ID No: 23);2-5/rh.51 (SEQ ID No: 22); 3-9/rh.52 (SEQ ID No: 18); 3-11/rh.53 (SEQ IDNO: 17); 5-3/rh.57 (SEQ ID No: 26); 5-22/rh.58 (SEQ ID No: 27);2-3/rh.61 (SEQ ID NO: 21); 4-8/rh.64 (SEQ ID No: 15); 3.1/hu.6 (SEQ IDNO: 5); 33.12/hu.17 (SEQ ID NO:4); 106.1/hu.37 (SEQ ID No: 10);LG-9/hu.39 (SEQ ID No: 24); 114.3/hu.40 (SEQ ID No: 11); 127.2/hu.41(SEQ ID NO:6); 127.5/hu.42 (SEQ ID No: 8); and hu.66 (SEQ ID NO: 173);2-15/rh.62 (SEQ ID NO: 33); 1-7/rh.48 (SEQ ID NO: 32); 4-9/rh.54 (SEQ IDNo: 40); 4-19/rh.55 (SEQ ID NO: 37); 52/hu.19 (SEQ ID NO: 62),52.1/hu.20 (SEQ ID NO: 63), 54.5/hu.23 (SEQ ID No: 60), 54.2/hu.22 (SEQID No: 67), 54.7/hu.24 (SEQ ID No: 66), 54.1/hu.21 (SEQ ID No: 65),54.4R/hu.27 (SEQ ID No: 64); 46.2/hu.28 (SEQ ID No: 68); 46.6/hu.29 (SEQID No: 69); 128.1/hu.43 (SEQ ID No: 80); 128.3/hu.44 (SEQ ID No: 81) and130.4/hu.48 (SEQ ID NO: 78); 3.1/hu.9 (SEQ ID No: 58); 16.8/hu.10 (SEQID No: 56); 16.12/hu.11 (SEQ ID No: 57); 145.1/hu.53 (SEQ ID No: 176);145.6/hu.55 (SEQ ID No: 178); 145.5/hu.54 (SEQ ID No: 177); 7.3/hu.7(SEQ ID No: 55); 52/hu.19 (SEQ ID No: 62); 33.4/hu.15 (SEQ ID No: 50);33.8/hu.16 (SEQ ID No: 51); 58.2/hu.25 (SEQ ID No: 49); 161.10/hu.60(SEQ ID No: 170); H-5/hu.3 (SEQ ID No: 44); H-1/hu.1 (SEQ ID No: 46);161.6/hu.61 (SEQ ID No: 174); hu.31 (SEQ ID No: 1); hu.32 (SEQ ID No:2); hu.46 (SEQ ID NO: 82); hu.34 (SEQ ID NO: 72); hu.47 (SEQ ID NO: 77);hu.63 (SEQ ID NO: 204); hu.56 (SEQ ID NO: 205); hu.45 (SEQ ID NO: 76);hu.57 (SEQ ID NO: 206); hu.35 (SEQ ID NO: 73); hu.58 (SEQ ID NO: 207);hu.51(SEQ ID NO: 208); hu.49 (SEQ ID NO: 209); hu.52 (SEQ ID NO: 210);hu.13 (SEQ ID NO: 71); hu.64 (SEQ ID NO: 212); rh.56 (SEQ ID NO: 54);hu.2 (SEQ ID NO: 48); hu.18 (SEQ ID NO: 52); hu.4 (SEQ ID NO: 47); andhu.67 (SEQ ID NO: 215). These sequences are also reproduced in theSequence Listing, which is incorporated by reference herein.

FIGS. 4A-4D provide an evaluation of gene transfer efficiency of novelprimate AAV-based vectors in vitro and in vivo. AAV vectors werepseudotyped as described [Gao et al, Proc Natl Acad Sci USA,99:11854-11859 (Sep. 3, 2002)] with capsids of AAVs 1, 2, 5, 7, 8 and 6and ch.5, rh.34, cy.5, rh.20, rh.8 and AAV9. For in vitro study, FIG.4A, 84-32 cells (293 cells expressing E4 of adenovirus serotypes) seededin a 95 well plate were infected with pseudotyped AAVCMVEGFP vectors atan MOI of 1×10⁴ GC per cell. Relative EGFP transduction efficiency wasestimated as percentage of green cells using a UV microscope at 48 hourspost-infection and shown on the Y axis. For in vivo study, the vectorsexpressing the secreted reporter gene A1AT were administered to theliver (FIG. 4B), lung (FIG. 4C) and muscle (FIG. 4D) of NCR nude mice(4-6 weeks old) at a dose of 1×10¹¹ GC per animal by intraportal (FIG.4B), intratracheal (FIG. 4C) and intramuscular injections (FIG. 4D),respectively. Serum A1AT levels (ng/mL) were compared at day 28 postgene transfer and presented on the Y axis. The X axis indicates the AAVsanalyzed and the clades to which they belong.

DETAILED DESCRIPTION OF THE INVENTION

In any arsenal of vectors useful in therapy or prophylaxis, a variety ofdistinct vectors capable of carrying a macromolecule to a target cell isdesirable, in order to permit selection of a vector source for a desiredapplication. To date, one of the concerns regarding the use of AAV asvectors was the lack of a variety of different virus sources. One way inwhich the present invention overcomes this problem is by providingclades of AAV, which are useful for selecting phylogenetically related,or where desired for a selected regimen, phylogenetically distinct, AAVand for predicting function. The invention further provides novel AAVviruses.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid, or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or anopen reading frame thereof, or another suitable fragment which is atleast 15 nucleotides in length. Examples of suitable fragments aredescribed herein.

The terms “sequence identity” “percent sequence identity” or “percentidentical” in the context of nucleic acid sequences refers to theresidues in the two sequences which are the same when aligned formaximum correspondence. The length of sequence identity comparison maybe over the full-length of the genome, the full-length of a gene codingsequence, or a fragment of at least about 500 to 5000 nucleotides, isdesired. However, identity among smaller fragments, e.g. of at leastabout nine nucleotides, usually at least about 20 to 24 nucleotides, atleast about 28 to 32 nucleotides, at least about 36 or more nucleotides,may also be desired. Similarly, “percent sequence identity” may bereadily determined for amino acid sequences, over the full-length of aprotein, or a fragment thereof. Suitably, a fragment is at least about 8amino acids in length, and may be up to about 700 amino acids. Examplesof suitable fragments are described herein.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid (or its complementary strand), there is aminoacid sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or aprotein thereof, e.g., a cap protein, a rep protein, or a fragmentthereof which is at least 8 amino acids, or more desirably, at least 15amino acids in length. Examples of suitable fragments are describedherein.

By the term “highly conserved” is meant at least 80% identity,preferably at least 90% identity, and more preferably, over 97%identity. Identity is readily determined by one of skill in the art byresort to algorithms and computer programs known by those of skill inthe art.

Generally, when referring to “identity”, “homology”, or “similarity”between two different adeno-associated viruses, “identity”, “homology”or “similarity” is determined in reference to “aligned” sequences.“Aligned” sequences or “alignments” refer to multiple nucleic acidsequences or protein (amino acids) sequences, often containingcorrections for missing or additional bases or amino acids as comparedto a reference sequence. In the examples, AAV alignments are performedusing the published AAV2 or AAV1 sequences as a reference point.However, one of skill in the art can readily select another AAV sequenceas a reference.

Alignments are performed using any of a variety of publicly orcommercially available Multiple Sequence Alignment Programs. Examples ofsuch programs include, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and“MEME”, which are accessible through Web Servers on the internet. Othersources for such programs are known to those of skill in the art.Alternatively, Vector NTI utilities are also used. There are also anumber of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta™, a program in GCG Version 6.1. Fasta™ providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta™ with its default parameters (a word size of 6 and the NOPAMfactor for the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Multiple sequence alignment programs are alsoavailable for amino acid sequences, e.g., the “Clustal X”, “MAP”,“PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs.Generally, any of these programs are used at default settings, althoughone of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

The term “serotype” is a distinction with respect to an AAV having acapsid which is serologically distinct from other AAV serotypes.Serologic distinctiveness is determined on the basis of the lack ofcross-reactivity between antibodies to the AAV as compared to other AAV.

Cross-reactivity is typically measured in a neutralizing antibody assay.For this assay polyclonal serum is generated against a specific AAV in arabbit or other suitable animal model using the adeno-associatedviruses. In this assay, the serum generated against a specific AAV isthen tested in its ability to neutralize either the same (homologous) ora heterologous AAV. The dilution that achieves 50% neutralization isconsidered the neutralizing antibody titer. If for two AAVs the quotientof the heterologous titer divided by the homologous titer is lower than16 in a reciprocal manner, those two vectors are considered as the sameserotype. Conversely, if the ratio of the heterologous titer over thehomologous titer is 16 or more in a reciprocal manner the two AAVs areconsidered distinct serotypes.

As defined herein, to form serotype 9, antibodies generated to aselected AAV capsid must not be cross-reactive with any of AAV 1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8. In one embodiment, the presentinvention provides an AAV capsid of a novel serotype, identified herein,as human AAV serotype 9.

As used throughout this specification and the claims, the terms“comprising” and “including” are inclusive of other components,elements, integers, steps and the like. Conversely, the term“consisting” and its variants are exclusive of other components,elements, integers, steps and the like.

I. Clades

In one aspect, the invention provides clades of AAV. A clade is a groupof AAV which are phylogenetically related to one another as determinedusing a Neighbor-Joining algorithm by a bootstrap value of at least 75%(of at least 1000 replicates) and a Poisson correction distancemeasurement of no more than 0.05, based on alignment of the AAV vp1amino acid sequence.

The Neighbor-Joining algorithm has been described extensively in theliterature. See, e.g., M. Nei and S. Kumar, Molecular Evolution andPhylogenetics (Oxford University Press, New York (2000). Computerprograms are available that can be used to implement this algorithm. Forexample, the MEGA v2.1 program implements the modified Nei-Gojoborimethod. Using these techniques and computer programs, and the sequenceof an AAV vp1 capsid protein, one of skill in the art can readilydetermine whether a selected AAV is contained in one of the cladesidentified herein, in another clade, or is outside these clades.

While the clades defined herein are based primarily upon naturallyoccurring AAV vp1 capsids, the clades are not limited to naturallyoccurring AAV. The clades can encompass non-naturally occurring AAV,including, without limitation, recombinant, modified or altered,chimeric, hybrid, synthetic, artificial, etc., AAV which arephylogenetically related as determined using a Neighbor-Joiningalgorithm at least 75% (of at least 1000 replicates) and a Poissoncorrection distance measurement of no more than 0.05, based on alignmentof the AAV vp1 amino acid sequence.

The clades described herein include Clade A (represented by AAV1 andAAV6), Clade B (represented by AAV2) and Clade C (represented by theAAV2-AAV3 hybrid), Clade D (represented by AAV7), Clade E (representedby AAV8), and Clade F (represented by human AAV9). These clades arerepresented by a member of the clade that is a previously described AAVserotype. Previously described AAV1 and AAV6 are members of a singleclade (Clade A) in which 4 isolates were recovered from 3 humans.Previously described AAV3 and AAV5 serotypes are clearly distinct fromone another, but were not detected in the screen described herein, andhave not been included in any of these clades.

Clade B (AAV2) and Clade C (the AAV2-AAV3 hybrid) are the most abundantof those found in humans (22 isolates from 12 individuals for AAV2 and17 isolates from 8 individuals for Clade C).

There are a large number of sequences grouped in either Clade D (AAV7)or Clade E (AAV8). Interestingly, both of these clades are prevalent indifferent species. Clade D is unique to rhesus and cynomologus macaqueswith 15 members being isolated from 10 different animals. Clade E isinteresting because it is found in both human and nonhuman primates: 9isolates were recovered from 7 humans and 21 isolates were obtained in 9different nonhuman primates including rhesus macaques, a baboon and apigtail monkey.

In two other animals the hybrid nature of certain sequences was proven,although all sequences in this case seem to have originated throughindividual and different recombinations of two co-infecting viruses (inboth animals a Clade D with a Clade E virus). None of these recombinantswere identified in other animals or subjects.

Since Clade C (the AAV2-AAV3 hybrid) clade was identified in 6 differenthuman subjects, the recombination event resulted in a fit progeny. Inthe case of the AAV7-AAV8 hybrids on the other hand, only fewconclusions can be drawn as to the implication of recombination in AAVevolution. These recombination events show that AAV is capable ofrecombining, thereby creating in-frame genes and in some casespackagable and/or infectious capsid structures. Clade C (the AAV2-AAV3hybrid clade) on the other hand is a group of viruses that has acquireda selective advantage through recombination that made them sustaincertain environmental pressures.

A. Clade A (Represented by AAV1 and AAV6):

In another aspect, the invention provides Clade A, which ischaracterized by containing the previously published AAV1 and AAV6. See,e.g., International Publication No. WO 00/28061, 18 May 2000; Rutledgeet al, J Virol, 72(1):309-319 (January 1998). In addition, this cladecontains novel AAV including, without limitation, 128.1/hu. 43 [SEQ IDNOs: 80 and 160]; 128.3/hu. 44 [SEQ ID Nos: 81 and 158]; 130.4/hu.48[SEQ ID NO: 78 and 157]; and hu.46 [SEQ ID NOs: 82 and 159]. Theinvention further provides a modified hu. 43 capsid [SEQ ID NO:236] anda modified hu. 46 capsid [SEQ ID NO:224].

In one embodiment, one or more of the members of this clade has a capsidwith an amino acid identity of at least 85% identity, at least 90%identity, at least 95% identity, or at least 97% identity over thefull-length of the vp1, the vp2, or the vp3 of the AAV1 and/or AAV6capsid.

In another embodiment, the invention provides novel AAV of Clade A,provided that none of the novel AAV comprises a capsid of any of AAV1 orAAV6. These AAV may include, without limitation, an AAV having a capsidderived from one or more of 128.1/hu. 43 [SEQ ID Nos: 80 and 160];modified hu.43 [SEQ ID NO:236] 128.3/hu. 44 [SEQ ID Nos: 81 and 158];hu.46 [SEQ ID NOs: 82 and 159]; modified hu. 46 [SEQ ID NO:224]; and130.4/hu.48 [SEQ ID NO: 78 and 157].

B. Clade B (AAV2 Clade):

In another embodiment, the invention provides a Clade B.

This clade is characterized by containing, at a minimum, the previouslydescribed AAV2 and novel AAV of the invention including, 52/hu.19 [SEQID NOs: 62 and 133], 52.1/hu.20 [SEQ ID NOs: 63 and 134], 54.5/hu.23[SEQ ID Nos: 60 and 137], 54.2/hu.22 [SEQ ID Nos: 67 and 138],54.7/hu.24 [SEQ ID Nos: 66 and 136], 54.1/hu.21 [SEQ ID Nos: 65 and135], 54.4R/hu.27 [SEQ ID Nos: 64 and 140]; 46.2/hu.28 [SEQ ID Nos: 68and 130]; 46.6/hu.29 [SEQ ID Nos: 69 and 132]; modified hu. 29 [SEQ IDNO: 225]; 172.1/hu.63 [SEQ ID NO: 171 and 195; GenBank Accession No.AY530624]; 172.2/hu. 64 [SEQ ID NO: 172 and 196; GenBank Accession No.AY530625]; 24.5/hu.13 [SEQ ID NO: 71 and 129; GenBank Accession No.AY530578]; 145.6/hu.56 [SEQ ID NO: 168 and 192]; hu.57 [SEQ ID Nos: 169and 193]; 136.1/hu.49 [SEQ ID NO: 165 and 189]; 156.1/hu.58 [SEQ ID NO:179 and 194]; 72.2/hu.34 [SEQ ID NO: 72 and 125; GenBank Accession No.AY530598]; 72.3/hu.35 [SEQ ID NO: 73 and 164; GenBank Accession No.AY530599]; 130.1/hu.47 [SEQ ID NO: 77 and 128]; 129.1/hu.45 (SEQ ID NO:76 and 127; GenBank Accession No. AY530608); 140.1/hu.51 [SEQ ID NO: 161and 190; GenBank Accession No. AY530613]; and 140.2/hu.52 [SEQ ID NO:167 and 191; GenBank Accession No. AY530614].

In one embodiment, one or more of the members of this clade has a capsidwith an amino acid identity of at least 85% identity, at least 90%identity, at least 95% identity, or at least 97% identity over thefull-length of the vp1, the vp2, or the vp3 of the AAV2 capsid.

In another embodiment, the invention provides novel AAV of Clade B,provided that none of the AAV has an AAV2 capsid. These AAV may include,without limitation, an AAV having a capsid derived from one or more ofthe following: 52/hu.19 [SEQ ID NOs: 62 and 133], 52.1/hu.20 [SEQ IDNOs: 63 and 134], 54.5/hu.23 [SEQ ID Nos: 60 and 137], 54.2/hu.22 [SEQID Nos: 67 and 138], 54.7/hu.24 [SEQ ID Nos: 66 and 136], 54.1/hu.21[SEQ ID Nos: 65 and 135], 54.4R/hu.27 [SEQ ID Nos: 64 and 140];46.2/hu.28 [SEQ ID Nos: 68 and 130]; 46.6/hu.29 [SEQ ID Nos: 69 and132]; modified hu. 29 [SEQ ID NO: 225]; 172.1/hu.63 [SEQ ID NO: 171 and195]; 172.2/hu. 64 [SEQ ID NO: 172 and 196]; 24.5/hu.13 [SEQ ID NO: 71and 129]; 145.6/hu.56 [SEQ ID NO: 168 and 192; GenBank Accession No.AY530618]; hu.57 [SEQ ID Nos: 169 and 193; GenBank Accession No.AY530619]; 136.1/hu.49 [SEQ ID NO: 165 and 189; GenBank Accession No.AY530612]; 156.1/hu.58 [SEQ ID NO: 179 and 194; GenBank Accession No.AY530620]; 72.2/hu.34 [SEQ ID NO: 72 and 125]; 72.3/hu.35 [SEQ ID NO: 73and 164]; 129.1/hu.45 [SEQ ID NO: 76 and 127]; 130.1/hu.47 [SEQ ID NO:77and 128; GenBank Accession No. AY530610]; 140.1/hu.51 [SEQ ID NO: 161and 190; GenBank Accession No. AY530613]; and 140.2/hu.52 [SEQ ID NO:167 and 191; GenBank Accession No. AY530614].

C. Clade C (AAV2-AAV3 Hybrid Clade)

In another aspect, the invention provides Clade C, which ischaracterized by containing AAV that are hybrids of the previouslypublished AAV2 and AAV3 such as H-6/hu.4; H-2/hu.2 [US PatentApplication 2003/0138772 (Jun. 24, 2003). In addition, this cladecontains novel AAV including, without limitation, 3.1/hu.9 [SEQ ID Nos:58 and 155]; 16.8/hu.10 [SEQ ID Nos: 56 and 156]; 16.12/hu.11 [SEQ IDNos: 57 and 153]; 145.1/hu.53 [SEQ ID Nos: 176 and 186]; 145.6/hu.55[SEQ ID Nos: 178 and 187]; 145.5/hu.54 [SEQ ID Nos: 177 and 188];7.3/hu.7 [SEQ ID Nos: 55 and 150; now deposited as GenBank Accession No.AY530628]; modified hu. 7 [SEQ ID NO: 226]; 33.4/hu.15 [SEQ ID Nos: 50and 147]; 33.8/hu.16 [SEQ ID Nos: 51 and 148]; hu.18 [SEQ ID NOs: 52 and149]; 58.2/hu.25 [SEQ ID Nos: 49 and 146]; 161.10/hu.60 [SEQ ID Nos: 170and 184]; H-5/hu.3 [SEQ ID Nos: 44 and 145]; H-1/hu.1 [SEQ ID Nos: 46and 144]; and 161.6/hu.61 [SEQ ID Nos: 174 and 185].

In one embodiment, one or more of the members of this clade has a capsidwith an amino acid identity of at least 85% identity, at least 90%identity, at least 95% identity, or at least 97% identity over thefull-length of the vp1, the vp2, or the vp3 of the hu.4 and/or hu.2capsid.

In another embodiment, the invention provides novel AAV of Clade C (theAAV2-AAV3 hybrid clade), provided that none of the novel AAV comprises acapsid of hu.2 or hu.4. These AAV may include, without limitation, anAAV having a capsid derived from one or more of 3.1/hu.9 [SEQ ID Nos: 58and 155]; 16.8/hu.10 [SEQ ID Nos: 56 and 156]; 16.12/hu.11 [SEQ ID Nos:57 and 153]; 145.1/hu.53 [SEQ ID Nos: 176 and 186]; 145.6/hu.55 [SEQ IDNos: 178 and 187]; 145.5/hu.54 [SEQ ID Nos: 177 and 188]; 7.3/hu.7 [SEQID Nos: 55 and 150]; modified hu.7 [SEQ ID NO:226]; 33.4/hu.15 [SEQ IDNos: 50 and 147]; 33.8/hu.16 [SEQ ID Nos: 51 and 148]; 58.2/hu.25 [SEQID Nos: 49 and 146]; 161.10/hu.60 [SEQ ID Nos: 170 and 184]; H-5/hu.3[SEQ ID Nos: 44 and 145]; H-1/hu.1 [SEQ ID Nos: 46 and 144]; and161.6/hu.61 [SEQ ID Nos: 174 and 185].

D. Clade D (AAV7 Clade)

In another embodiment, the invention provides Clade D. This clade ischaracterized by containing the previously described AAV7 [G. Gao et al,Proc. Natl Acad. Sci USA, 99:11854-9 (Sep. 3, 2002). The nucleic acidsequences encoding the AAV7 capsid are reproduced in SEQ ID NO: 184; theamino acid sequences of the AAV7 capsid are reproduced in SEQ ID NO:185. In addition, the clade contains a number of previously describedAAV sequences, including: cy.2; cy.3; cy.4; cy.5; cy.6; rh.13; rh.37;rh. 36; and rh.35 [US Published Patent Application No. US 2003/0138772A1 (Jul. 24, 2003)]. Additionally, the AAV7 clade contains novel AAVsequences, including, without limitation, 2-15/rh.62 [SEQ ID Nos: 33 and114]; 1-7/rh.48 [SEQ ID Nos: 32 and 115]; 4-9/rh.54 [SEQ ID Nos: 40 and116]; and 4-19/rh.55 [SEQ ID Nos: 37 and 117]. The invention furtherincludes modified cy. 5 [SEQ ID NO: 227]; modified rh.13 [SEQ ID NO:228]; and modified rh. 37 [SEQ ID NO: 229].

In one embodiment, one or more of the members of this clade has a capsidwith an amino acid identity of at least 85% identity, at least 90%identity, at least 95% identity, or at least 97% identity over thefull-length of the vp1, the vp2, or the vp3 of the AAV7 capsid, SEQ IDNO: 184 and 185.

In another embodiment, the invention provides novel AAV of Clade D,provided that none of the novel AAV comprises a capsid of any of cy.2;cy.3; cy.4; cy.5; cy.6; rh.13; rh.37; rh. 36; and rh.35. These AAV mayinclude, without limitation, an AAV having a capsid derived from one ormore of the following 2-15/rh.62 [SEQ ID Nos: 33 and 114]; 1-7/rh.48[SEQ ID Nos: 32 and 115]; 4-9/rh.54 [SEQ ID Nos: 40 and 116]; and4-19/rh.55 [SEQ ID Nos: 37 and 117].

E. Clade E (AAV8 Clade)

In one aspect, the invention provides Clade E. This clade ischaracterized by containing the previously described AAV8 [G. Gao et al,Proc. Natl Acad. Sci USA, 99:11854-9 (Sep. 3, 2002)], 43.1/rh.2;44.2/rh.10; rh. 25; 29.3/bb.1; and 29.5/bb.2 [US Published PatentApplication No. US 2003/0138772 A1 (Jul. 24, 2003)].

Further, the clade novel AAV sequences, including, without limitation,including, e.g., 30.10/pi.1 [SEQ ID NOs: 28 and 93], 30.12/pi.2 [SEQ IDNOs: 30 and 95, 30.19/pi.3 [SEQ ID NOs: 29 and 94], LG-4/rh.38 [SEQ IDNos: 7 and 86]; LG-10/rh.40 [SEQ ID Nos: 14 and 92]; N721-8/rh.43 [SEQID Nos: 43 and 163]; 1-8/rh.49 [SEQ ID NOs: 25 and 103]; 2-4/rh.50 [SEQID Nos: 23 and 108]; 2-5/rh.51 [SEQ ID Nos: 22 and 104]; 3-9/rh.52 [SEQID Nos: 18 and 96]; 3-11/rh.53 [SEQ ID NOs: 17 and 97]; 5-3/rh.57 [SEQID Nos: 26 and 105]; 5-22/rh.58 [SEQ ID Nos: 27 and 58]; 2-3/rh.61 [SEQID NOs: 21 and 107]; 4-8/rh.64 [SEQ ID Nos: 15 and 99]; 3.1/hu.6 [SEQ IDNO: 5 and 84]; 33.12/hu.17 [SEQ ID NO:4 and 83]; 106.1/hu.37 [SEQ IDNos: 10 and 88]; LG-9/hu.39 [SEQ ID Nos: 24 and 102]; 114.3/hu. 40 [SEQID Nos: 11 and 87]; 127.2/hu.41 [SEQ ID NO:6 and 91]; 127.5/hu.42 [SEQID Nos: 8 and 85]; hu. 66 [SEQ ID NOs: 173 and 197]; and hu.67 [SEQ IDNOs: 174 and 198]. This clade further includes modified rh. 2 [SEQ IDNO: 231]; modified rh. 58 [SEQ ID NO: 232]; modified rh. 64 [SEQ ID NO:233].

In one embodiment, one or more of the members of this clade has a capsidwith an amino acid identity of at least 85% identity, at least 90%identity, at least 95% identity, or at least 97% identity over thefull-length of the vp1, the vp2, or the vp3 of the AAV8 capsid. Thenucleic acid sequences encoding the AAV8 capsid are reproduced in SEQ IDNO: 186 and the amino acid sequences of the capsid are reproduced in SEQID NO:187.

In another embodiment, the invention provides novel AAV of Clade E,provided that none of the novel AAV comprises a capsid of any of AAV8,rh.8; 44.2/rh.10; rh. 25; 29.3/bb.1; and 29.5/bb.2 [US Published PatentApplication No. US 2003/0138772 A1 (Jul. 24, 2003)]. These AAV mayinclude, without limitation, an AAV having a capsid derived from one ormore of the following: 30.10/pi.1 [SEQ ID NOs:28 and 93], 30.12/pi.2[SEQ ID NOs:30 and 95, 30.19/pi.3 [SEQ ID NOs:29 and 94], LG-4/rh.38[SEQ ID Nos: 7 and 86]; LG-10/rh.40 [SEQ ID Nos: 14 and 92];N721-8/rh.43 [SEQ ID Nos: 43 and 163]; 1-8/rh.49 [SEQ ID NOs: 25 and103]; 2-4/rh.50 [SEQ ID Nos: 23 and 108]; 2-5/rh.51 [SEQ ID Nos: 22 and104]; 3-9/rh.52 [SEQ ID Nos: 18 and 96]; 3-11/rh.53 [SEQ ID NOs: 17 and97]; 5-3/rh.57 [SEQ ID Nos: 26 and 105]; 5-22/rh.58 [SEQ ID Nos: 27 and58]; modified rh. 58 [SEQ ID NO: 232]; 2-3/rh.61 [SEQ ID NOs: 21 and107]; 4-8/rh.64 [SEQ ID Nos: 15 and 99]; modified rh. 64[SEQ ID NO:233]; 3.1/hu.6 [SEQ ID NO: 5 and 84]; 33.12/hu.17 [SEQ ID NO:4 and 83];106.1/hu.37 [SEQ ID Nos: 10 and 88]; LG-9/hu.39 [SEQ ID Nos: 24 and102]; 114.3/hu. 40 [SEQ ID Nos: 11 and 87]; 127.2/hu.41 [SEQ ID NO:6 and91]; 127.5/hu.42 [SEQ ID Nos: 8 and 85]; hu. 66 [SEQ ID NOs: 173 and197]; and hu.67 [SEQ ID NOs: 174 and 198].

F. Clade F (AAV 9 Clade)

This clade is identified by the name of a novel AAV serotype identifiedherein as hu.14/AAV9 [SEQ ID Nos: 3 and 123]. In addition, this cladecontains other novel sequences including, hu.31 [SEQ ID NOs:1 and 121];and hu.32 [SEQ ID Nos: 2 and 122].

In one embodiment, one or more of the members of this clade has a capsidwith an amino acid identity of at least 85% identity, at least 90%identity, at least 95% identity, or at least 97% identity over thefull-length of the vp1, the vp2, or the vp3 of the AAV9 capsid, SEQ IDNO: 3 and 123.

In another embodiment, the invention provides novel AAV of Clade F,which include, without limitation, an AAV having a capsid derived fromone or more of hu.14/AAV9 [SEQ ID Nos: 3 and 123], hu.31 [SEQ ID NOs:1and 121] and hu.32 [SEQ ID Nos: 1 and 122].

The AAV clades of the invention are useful for a variety of purposes,including providing ready collections of related AAV for generatingviral vectors, and for generating targeting molecules. These clades mayalso be used as tools for a variety of purposes that will be readilyapparent to one of skill in the art.

II. Novel AAV Sequences

The invention provides the nucleic acid sequences and amino acids of anovel AAV serotype, which is termed interchangeably herein as clonehu.14/28.4 and huAAV9. These sequences are useful for constructingvectors that are highly efficient in transduction of liver, muscle andlung. This novel AAV and its sequences are also useful for a variety ofother purposes. These sequences are being submitted with GenBank andhave been assigned the accession numbers identified herein.

The invention further provides the nucleic acid sequences and amino acidsequences of a number of novel AAV. Many of these sequence include thosedescribed above as members of a clade, as summarized below.

128.1/hu. 43 [SEQ ID Nos: 80 and 160 GenBank Accession No. AY530606];modified hu. 43 [SEQ ID NO:236]; 128.3/hu. 44 [SEQ ID Nos: 81 and 158;GenBank Accession No. AY530607] and 130.4/hu.48 [SEQ ID NO: 78 and 157;GenBank Accession No. AY530611]; from the Clade A;

52/hu.19 [SEQ ID NOs: 62 and 133; GenBank Accession No. AY530584],52.1/hu.20 [SEQ ID NOs: 63 and 134; GenBank Accession No. AY530586],54.5/hu.23 [SEQ ID Nos: 60 and 137; GenBank Accession No. AY530589],54.2/hu.22 [SEQ ID Nos: 67 and 138; GenBank Accession No. AY530588],54.7/hu.24 [SEQ ID Nos: 66 and 136; GenBank Accession No. AY530590],54.1/hu.21 [SEQ ID Nos: 65 and 135; GenBank Accession No. AY530587],54.4R/hu.27 [SEQ ID Nos: 64 and 140; GenBank Accession No. AY530592];46.2/hu.28 [SEQ ID Nos: 68 and 130; GenBank Accession No. AY530593];46.6/hu.29 [SEQ ID Nos: 69 and 132; GenBank Accession No. AY530594];modified hu. 29 [SEQ ID NO: 225]; 172.1/hu.63 [SEQ ID NO: 171 and 195];and 140.2/hu.52 (SEQ ID NO: 167 and 191; from Clade B;

3.1/hu.9 [SEQ ID Nos: 58 and 155; GenBank Accession No. AY530626];16.8/hu.10 [SEQ ID Nos: 56 and 156; GenBank Accession No. AY530576];16.12/hu.11 [SEQ ID Nos: 57 and 153; GenBank Accession No. AY530577];145.1/hu.53 [SEQ ID Nos: 176 and 186; GenBank Accession No. AY530615];145.6/hu.55 [SEQ ID Nos: 178 and 187; GenBank Accession No. AY530617];145.5/hu.54 [SEQ ID Nos: 177 and 188; GenBank Accession No. AY530616];7.3/hu.7 [SEQ ID Nos: 55 and 150; GenBank Accession No. AY530628];modified hu. 7 [SEQ ID NO: 226]; hu.18 [SEQ ID Nos: 52 and 149; GenBankAccession No. AY530583]; 33.4/hu.15 [SEQ ID Nos: 50 and 147; GenBankAccession No. AY530580]; 33.8/hu.16 [SEQ ID Nos: 51 and 148; GenBankAccession No. AY530581]; 58.2/hu.25 [SEQ ID Nos: 49 and 146; GenBankAccession No. AY530591]; 161.10/hu.60 [SEQ ID Nos: 170 and 184; GenBankAccession No. AY530622]; H-5/hu.3 [SEQ ID Nos: 44 and 145; GenBankAccession No. AY530595]; H-1/hu.1 [SEQ ID Nos: 46 and 144; GenBankAccession No. AY530575]; and 161.6/hu.61 [SEQ ID Nos: 174 and 185;GenBank Accession No. AY530623] from Clade C;

2-15/rh.62 [SEQ ID Nos: 33 and 114; GenBank Accession No. AY530573];1-7/rh.48 [SEQ ID Nos: 32 and 115; GenBank Accession No. AY530561];4-9/rh.54 [SEQ ID Nos: 40 and 116; GenBank Accession No. AY530567]; and4-19/rh.55 [SEQ ID Nos: 37 and 117; GenBank Accession No. AY530568];modified cy. 5 [SEQ ID NO: 227]; modified rh.13 [SEQ ID NO: 228]; andmodified rh. 37 [SEQ ID NO: 229] from the Clade D;

30.10/pi.1 [SEQ ID NOs:28 and 93; GenBank Accession No. AY53055],30.12/pi.2 [SEQ ID NOs:30 and 95; GenBank Accession No. AY 530554],30.19/pi.3 [SEQ ID NOs:29 and 94; GenBank Accession No. AY530555],LG-4/rh.38 [SEQ ID Nos: 7 and 86; GenBank Accession No. AY 530558];LG-10/rh.40 [SEQ ID Nos: 14 and 92; GenBank Accession No. AY530559];N721-8/rh.43 [SEQ ID Nos: 43 and 163; GenBank Accession No. AY530560];1-8/rh.49 [SEQ ID NOs: 25 and 103; GenBank Accession No. AY530561];2-4/rh.50 [SEQ ID Nos: 23 and 108; GenBank Accession No. AY530563];2-5/rh.51 [SEQ ID Nos: 22 and 104; GenBank Accession No. 530564];3-9/rh.52 [SEQ ID Nos: 18 and 96; GenBank Accession No. AY530565];3-11/rh.53 [SEQ ID Nos: 17 and 97; GenBank Accession No. AY530566];5-3/rh.57 [SEQ ID Nos: 26 and 105; GenBank Accession No. AY530569];5-22/rh.58 [SEQ ID Nos: 27 and 58; GenBank Accession No. 530570];modified rh. 58 [SEQ ID NO: 232]; 2-3/rh.61 [SEQ ID Nos: 21 and 107;GenBank Accession No. AY530572]; 4-8/rh.64 [SEQ ID Nos: 15 and 99;GenBank Accession No. AY530574]; modified rh. 64[SEQ ID NO: 233];3.1/hu.6 [SEQ ID NO: 5 and 84; GenBank Accession No. AY530621];33.12/hu.17 [SEQ ID NO:4 and 83; GenBank Accession No. AY530582];106.1/hu.37 [SEQ ID Nos: 10 and 88; GenBank Accession No. AY530600];LG-9/hu.39 [SEQ ID Nos: 24 and 102; GenBank Accession No. AY530601];114.3/hu. 40 [SEQ ID Nos: 11 and 87; GenBank Accession No. AY530603];127.2/hu.41 [SEQ ID NO:6 and 91; GenBank Accession No. AY530604];127.5/hu.42 [SEQ ID Nos: 8 and 85; GenBank Accession No. AY530605]; andhu. 66 [SEQ ID NOs: 173 and 197; GenBank Accession No. AY530626]; andhu.67 [SEQ ID NOs: 174 and 198; GenBank Accession No. AY530627]; andmodified rh.2 [SEQ ID NO:231]; from Clade E;

hu.14/AAV9 [SEQ ID Nos: 3 and 123; GenBank Accession No. AY530579],hu.31 [SEQ ID NOs:1 and 121; AY530596] and hu.32 [SEQ ID Nos: 1 and 122;GenBank Accession No. AY530597] from Clade F.

In addition, the present invention provides AAV sequences, including,rh.59 [SEQ ID NO: 49 and 110]; rh.60 [SEQ ID NO: 31 and 120; GenBankAccession No. AY530571], modified ch.5 [SEQ ID NO: 234]; and modifiedrh. 8 [SEQ ID NO: 235], which are outside the definition of the cladesdescribed above.

Also provided are fragments of the AAV sequences of the invention. Eachof these fragments may be readily utilized in a variety of vectorsystems and host cells. Among desirable AAV fragments are the capproteins, including the vp1, vp2, vp3 and hypervariable regions. Wheredesired, the methodology described in published US Patent PublicationNo. US 2003/0138772 A1 (Jul. 24, 2003)] can be used to obtain the repsequences for the AAV clones identified above. Such rep sequencesinclude, e.g., rep 78, rep 68, rep 52, and rep 40, and the sequencesencoding these proteins. Similarly, other fragments of these clones maybe obtained using the techniques described in the referenced patentpublication, including the AAV inverted terminal repeat (ITRs), AAV P19sequences, AAV P40 sequences, the rep binding site, and the terminalresolute site (TRS). Still other suitable fragments will be readilyapparent to those of skill in the art.

The capsid and other fragments of the invention can be readily utilizedin a variety of vector systems and host cells. Such fragments may beused alone, in combination with other AAV sequences or fragments, or incombination with elements from other AAV or non-AAV viral sequences. Inone particularly desirable embodiment, a vector contains the AAV capand/or rep sequences of the invention.

The AAV sequences and fragments thereof are useful in production ofrAAV, and are also useful as antisense delivery vectors, gene therapyvectors, or vaccine vectors. The invention further provides nucleic acidmolecules, gene delivery vectors, and host cells which contain the AAVsequences of the invention.

Suitable fragments can be determined using the information providedherein.

As described herein, the vectors of the invention containing the AAVcapsid proteins of the invention are particularly well suited for use inapplications in which the neutralizing antibodies diminish theeffectiveness of other AAV serotype based vectors, as well as otherviral vectors. The rAAV vectors of the invention are particularlyadvantageous in rAAV readministration and repeat gene therapy.

These and other embodiments and advantages of the invention aredescribed in more detail below.

A. AAV Serotype 9/hu14 Sequences

The invention provides the nucleic acid sequences and amino acids of anovel AAV, which is termed interchangeable herein as clone hu.14(formerly termed 28.4) and huAAV9. As defined herein, novel serotypeAAV9 refers to AAV having a capsid which generates antibodies whichcross-react serologically with the capsid having the sequence of hu. 14[SEQ ID NO: 123] and which antibodies do not cross-react serologicallywith antibodies generated to the capsids of any of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7 or AAV8.

1. Nucleic Acid Sequences

The AAV9 nucleic acid sequences of the invention include the DNAsequences of SEQ ID NO: 3, which consists of 2211 nucleotides.

The nucleic acid sequences of the invention further encompass the strandwhich is complementary to SEQ ID NO: 3, as well as the RNA and cDNAsequences corresponding to SEQ ID NO: 3, and its complementary strand.Also included in the nucleic acid sequences of the invention are naturalvariants and engineered modifications of SEQ ID NO: 3 and itscomplementary strand. Such modifications include, for example, labelsthat are known in the art, methylation, and substitution of one or moreof the naturally occurring nucleotides with a degenerate nucleotide.

Further included in this invention are nucleic acid sequences which aregreater than about 90%, more preferably at least about 95%, and mostpreferably at least about 98 to 99%, identical or homologous to SEQ IDNO: 3.

Also included within the invention are fragments of SEQ ID NO: 3, itscomplementary strand, and cDNA and RNA complementary thereto. Suitablefragments are at least 15 nucleotides in length, and encompassfunctional fragments, i.e., fragments which are of biological interest.Such fragments include the sequences encoding the three variableproteins (vp) of the AAV9/HU.14 capsid which are alternative splicevariants: vp1 [nt 1 to 2211 of SEQ ID NO:3]; vp2 [about nt 411 to 2211of SEQ ID NO:3]; and vp 3 [about nt 609 to 2211 of SEQ ID NO:3]. Othersuitable fragments of SEQ ID NO: 3, include the fragment which containsthe start codon for the AAV9/HU.14 capsid protein, and the fragmentsencoding the hypervariable regions of the vp1 capsid protein, which aredescribed herein.

In addition to including the nucleic acid sequences provided in thefigures and Sequence Listing, the present invention includes nucleicacid molecules and sequences which are designed to express the aminoacid sequences, proteins and peptides of the AAV serotypes of theinvention. Thus, the invention includes nucleic acid sequences whichencode the following novel AAV amino acid sequences and artificial AAVserotypes generated using these sequences and/or unique fragmentsthereof.

As used herein, artificial AAV serotypes include, without limitation,AAVs with a non-naturally occurring capsid protein. Such an artificialcapsid may be generated by any suitable technique, using a novel AAVsequence of the invention (e.g., a fragment of a vp1 capsid protein) incombination with heterologous sequences which may be obtained fromanother AAV serotype (known or novel), non-contiguous portions of thesame AAV serotype, from a non-AAV viral source, or from a non-viralsource. An artificial AAV serotype may be, without limitation, achimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAVcapsid.

2. HU.14/AAV9 Amino Acid Sequences, Proteins and Peptides

The invention further provides proteins and fragments thereof which areencoded by the hu.14/AAV9 nucleic acids of the invention, and hu.14/AAV9proteins and fragments which are generated by other methods. As usedherein, these proteins include the assembled capsid. The inventionfurther encompasses AAV serotypes generated using sequences of the novelAAV serotype of the invention, which are generated using synthetic,recombinant or other techniques known to those of skill in the art. Theinvention is not limited to novel AAV amino acid sequences, peptides andproteins expressed from the novel AAV nucleic acid sequences of theinvention, but encompasses amino acid sequences, peptides and proteinsgenerated by other methods known in the art, including, e.g., bychemical synthesis, by other synthetic techniques, or by other methods.The sequences of any of the AAV capsids provided herein can be readilygenerated using a variety of techniques.

Suitable production techniques are well known to those of skill in theart. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively,peptides can also be synthesized by the well-known solid phase peptidesynthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962);Stewart and Young, Solid Phase Peptide Synthesis (Freeman, SanFrancisco, 1969) pp. 27-62). These and other suitable production methodsare within the knowledge of those of skill in the art and are not alimitation of the present invention.

Particularly desirable proteins include the AAV capsid proteins, whichare encoded by the nucleotide sequences identified above. The AAV capsidis composed of three proteins, vp1, vp2 and vp3, which are alternativesplice variants. The full-length sequence provided in FIG. 2 is that ofvp1. The AAV9/HU.14 capsid proteins include vp1 [amino acids (aa) 1 to736 of SEQ ID NO: 123], vp2 [about aa 138 to 736 of SEQ ID NO: 123], vp3[about aa 203 to 736 of SEQ ID NO: 123], and functional fragmentsthereof. Other desirable fragments of the capsid protein include theconstant and variable regions, located between hypervariable regions(HVR). Other desirable fragments of the capsid protein include the HVRthemselves.

An algorithm developed to determine areas of sequence divergence in AAV2has yielded 12 hypervariable regions (HVR) of which 5 overlap or arepart of the four previously described variable regions. [Chiorini et al,J. Virol, 73:1309-19 (1999); Rutledge et al, J. Virol., 72:309-319]Using this algorithm and/or the alignment techniques described herein,the HVR of the novel AAV serotypes are determined. For example, the HVRare located as follows: HVR1, aa 146-152; HVR2, aa 182-186; HVR3, aa262-264; HVR4, aa 381-383; HVR5, aa 450-474; HVR6, aa 490-495; HVR7, aa500-504; HVR8, aa 514-522; HVR9, aa 534-555; HVR10, aa 581-594; HVR11,aa 658-667; and HVR12, aa 705-719 [the numbering system is based on analignment which uses the AAV2 vp1 as a point of reference]. Using thealignment provided herein performed using the Clustal X program atdefault settings, or using other commercially or publicly availablealignment programs at default settings such as are described herein, oneof skill in the art can readily determine corresponding fragments of thenovel AAV capsids of the invention.

Still other desirable fragments of the AAV9/HU.14 capsid protein includeamino acids 1 to 184 of SEQ ID NO: 123, amino acids 199 to 259; aminoacids 274 to 446; amino acids 603 to 659; amino acids 670 to 706; aminoacids 724 to 736 of SEQ ID NO: 123; aa 185-198; aa 260-273; aa447-477;aa495-602; aa660-669; and aa707-723. Additionally, examples of othersuitable fragments of AAV capsids include, with respect to the numberingof AAV9 [SEQ ID NO: 123], aa 24-42, aa 25-28; aa 81-85; aa133-165; aa134-165; aa 137-143; aa 154-156; aa 194-208; aa 261-274; aa 262-274; aa171-173; aa 413-417; aa 449-478; aa 494-525; aa 534-571; aa 581-601; aa660-671; aa 709-723. Using the alignment provided herein performed usingthe Clustal X program at default settings, or using other commerciallyor publicly available alignment programs at default settings, one ofskill in the art can readily determine corresponding fragments of thenovel AAV capsids of the invention.

Still other desirable AAV9/HU.14 proteins include the rep proteinsinclude rep68/78 and rep40/52.

Suitably, fragments are at least 8 amino acids in length. However,fragments of other desired lengths may be readily utilized. Suchfragments may be produced recombinantly or by other suitable means,e.g., chemical synthesis.

The invention further provides other AAV9/HU.14 sequences which areidentified using the sequence information provided herein. For example,given the AAV9/HU.14 sequences provided herein, infectious AAV9/HU.14may be isolated using genome walking technology (Siebert et al., 1995,Nucleic Acid Research, 23:1087-1088, Friezner-Degen et al., 1986, J.Biol. Chem. 261:6972-6985, BD Biosciences Clontech, Palo Alto, Calif.).Genome walking is particularly well suited for identifying and isolatingthe sequences adjacent to the novel sequences identified according tothe method of the invention. This technique is also useful for isolatinginverted terminal repeat (ITRs) of the novel AAV9/HU.14 serotype, basedupon the novel AAV capsid and rep sequences provided herein.

The sequences, proteins, and fragments of the invention may be producedby any suitable means, including recombinant production, chemicalsynthesis, or other synthetic means. Such production methods are withinthe knowledge of those of skill in the art and are not a limitation ofthe present invention.

III. Production of rAAV with Novel AAV Capsids

The invention encompasses novel AAV capsid sequences of which are freeof DNA and/or cellular material with these viruses are associated innature. To avoid repeating all of the novel AAV capsids provided herein,reference is made throughout this and the following sections to thehu.14/AAV9 capsid. However, it should be appreciated that the othernovel AAV capsid sequences of the invention can be used in a similarmanner.

In another aspect, the present invention provides molecules that utilizethe novel AAV sequences of the invention, including fragments thereof,for production of molecules useful in delivery of a heterologous gene orother nucleic acid sequences to a target cell.

In another aspect, the present invention provides molecules that utilizethe AAV sequences of the invention, including fragments thereof, forproduction of viral vectors useful in delivery of a heterologous gene orother nucleic acid sequences to a target cell.

The molecules of the invention which contain AAV sequences include anygenetic element (vector) which may be delivered to a host cell, e.g.,naked DNA, a plasmid, phage, transposon, cosmid, episome, a protein in anon-viral delivery vehicle (e.g., a lipid-based carrier), virus, etc.,which transfers the sequences carried thereon. The selected vector maybe delivered by any suitable method, including transfection,electroporation, liposome delivery, membrane fusion techniques, highvelocity DNA-coated pellets, viral infection and protoplast fusion. Themethods used to construct any embodiment of this invention are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.

In one embodiment, the vectors of the invention contain, inter alia,sequences encoding an AAV capsid of the invention or a fragment thereof.In another embodiment, the vectors of the invention contain, at aminimum, sequences encoding an AAV rep protein or a fragment thereof.Optionally, vectors of the invention may contain both AAV cap and repproteins. In vectors in which both AAV rep and cap are provided, the AAVrep and AAV cap sequences can originate from an AAV of the same clade.Alternatively, the present invention provides vectors in which the repsequences are from an AAV source which differs from that which isproviding the cap sequences. In one embodiment, the rep and capsequences are expressed from separate sources (e.g., separate vectors,or a host cell and a vector). In another embodiment, these rep sequencesare fused in frame to cap sequences of a different AAV source to form achimeric AAV vector. Optionally, the vectors of the invention arevectors packaged in an AAV capsid of the invention. These vectors andother vectors described herein can further contain a minigene comprisinga selected transgene which is flanked by AAV 5′ ITR and AAV 3′ ITR.

Thus, in one embodiment, the vectors described herein contain nucleicacid sequences encoding an intact AAV capsid which may be from a singleAAV sequence (e.g., AAV9/HU.14). Such a capsid may comprise amino acids1 to 736 of SEQ ID NO:123. Alternatively, these vectors containsequences encoding artificial capsids which contain one or morefragments of the AAV9/HU.14 capsid fused to heterologous AAV or non-AAVcapsid proteins (or fragments thereof). These artificial capsid proteinsare selected from non-contiguous portions of the AAV9/HU.14 capsid orfrom capsids of other AAVs. For example, a rAAV may have a capsidprotein comprising one or more of the AAV9/HU.14 capsid regions selectedfrom the vp2 and/or vp3, or from vp 1, or fragments thereof selectedfrom amino acids 1 to 184, amino acids 199 to 259; amino acids 274 to446; amino acids 603 to 659; amino acids 670 to 706; amino acids 724 to738 of the AAV9/HU.14 capsid, SEQ ID NO: 123. In another example, it maybe desirable to alter the start codon of the vp3 protein to GTG.Alternatively, the rAAV may contain one or more of the AAV serotype 9capsid protein hypervariable regions which are identified herein, orother fragment including, without limitation, aa 185-198; aa 260-273;aa447-477; aa495-602; aa660-669; and aa707-723 of the AAV9/HU.14 capsid.See, SEQ ID NO: 123. These modifications may be to increase expression,yield, and/or to improve purification in the selected expressionsystems, or for another desired purpose (e.g., to change tropism oralter neutralizing antibody epitopes).

The vectors described herein, e.g., a plasmid, are useful for a varietyof purposes, but are particularly well suited for use in production of arAAV containing a capsid comprising AAV sequences or a fragment thereof.These vectors, including rAAV, their elements, construction, and usesare described in detail herein.

In one aspect, the invention provides a method of generating arecombinant adeno-associated virus (AAV) having an AAV serotype 9capsid, or a portion thereof. Such a method involves culturing a hostcell which contains a nucleic acid sequence encoding an AAV serotype 9capsid protein, or fragment thereof, as defined herein; a functional repgene; a minigene composed of, at a minimum, AAV inverted terminalrepeats (ITRs) and a transgene; and sufficient helper functions topermit packaging of the minigene into the AAV9/HU.14 capsid protein.

The components required to be cultured in the host cell to package anAAV minigene in an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,minigene, rep sequences, cap sequences, and/or helper functions) may beprovided by a stable host cell which has been engineered to contain oneor more of the required components using methods known to those of skillin the art. Most suitably, such a stable host cell will contain therequired component(s) under the control of an inducible promoter.However, the required component(s) may be under the control of aconstitutive promoter. Examples of suitable inducible and constitutivepromoters are provided herein, in the discussion of regulatory elementssuitable for use with the transgene. In still another alternative, aselected stable host cell may contain selected component(s) under thecontrol of a constitutive promoter and other selected component(s) underthe control of one or more inducible promoters. For example, a stablehost cell may be generated which is derived from 293 cells (whichcontain E1 helper functions under the control of a constitutivepromoter), but which contains the rep and/or cap proteins under thecontrol of inducible promoters. Still other stable host cells may begenerated by one of skill in the art.

The minigene, rep sequences, cap sequences, and helper functionsrequired for producing the rAAV of the invention may be delivered to thepackaging host cell in the form of any genetic element which transferthe sequences carried thereon. The selected genetic element may bedelivered by any suitable method, including those described herein. Themethods used to construct any embodiment of this invention are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods ofgenerating rAAV virions are well known and the selection of a suitablemethod is not a limitation on the present invention. See, e.g., K.Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

Unless otherwise specified, the AAV ITRs, and other selected AAVcomponents described herein, may be readily selected from among any AAV,including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV9 and one of the other novel AAV sequences of the invention. TheseITRs or other AAV components may be readily isolated using techniquesavailable to those of skill in the art from an AAV sequence. Such AAVmay be isolated or obtained from academic, commercial, or public sources(e.g., the American Type Culture Collection, Manassas, Va.).Alternatively, the AAV sequences may be obtained through synthetic orother suitable means by reference to published sequences such as areavailable in the literature or in databases such as, e.g., GenBank®,PubMed®, or the like.

A. The Minigene

The minigene is composed of, at a minimum, a transgene and itsregulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). In one desirable embodiment, the ITRs of AAV serotype 2 areused. However, ITRs from other suitable sources may be selected. It isthis minigene that is packaged into a capsid protein and delivered to aselected host cell.

1. The Transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),enhanced GFP (EGFP), chloramphenicol acetyltransferase (CAT),luciferase, membrane bound proteins including, for example, CD2, CD4,CD8, the influenza hemagglutinin protein, and others well known in theart, to which high affinity antibodies directed thereto exist or can beproduced by conventional means, and fusion proteins comprising amembrane bound protein appropriately fused to an antigen tag domainfrom, among others, hemagglutinin or Myc.

These coding sequences, when associated with regulatory elements whichdrive their expression, provide signals detectable by conventionalmeans, including enzymatic, radiographic, colorimetric, fluorescence orother spectrographic assays, fluorescent activating cell sorting assaysand immunological assays, including enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example,where the marker sequence is the LacZ gene, the presence of the vectorcarrying the signal is detected by assays for beta-galactosidaseactivity. Where the transgene is green fluorescent protein orluciferase, the vector carrying the signal may be measured visually bycolor or light production in a luminometer.

However, desirably, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, dominant negative mutants, or catalytic RNAs.Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalyticRNAs, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNAs.One example of a useful RNA sequence is a sequence which inhibits orextinguishes expression of a targeted nucleic acid sequence in thetreated animal. Typically, suitable target sequences include oncologictargets and viral diseases. See, for examples of such targets theoncologic targets and viruses identified below in the section relatingto immunogens.

The transgene may be used to correct or ameliorate gene deficiencies,which may include deficiencies in which normal genes are expressed atless than normal levels or deficiencies in which the functional geneproduct is not expressed. Alternatively, the transgene may provide aproduct to a cell which is not natively expressed in the cell type or inthe host. A preferred type of transgene sequence encodes a therapeuticprotein or polypeptide which is expressed in a host cell. The inventionfurther includes using multiple transgenes. In certain situations, adifferent transgene may be used to encode each subunit of a protein, orto encode different peptides or proteins. This is desirable when thesize of the DNA encoding the protein subunit is large, e.g., for animmunoglobulin, the platelet-derived growth factor, or a dystrophinprotein. In order for the cell to produce the multi-subunit protein, acell is infected with the recombinant virus containing each of thedifferent subunits. Alternatively, different subunits of a protein maybe encoded by the same transgene. In this case, a single transgeneincludes the DNA encoding each of the subunits, with the DNA for eachsubunit separated by an internal ribozyme entry site (IRES). This isdesirable when the size of the DNA encoding each of the subunits issmall, e.g., the total size of the DNA encoding the subunits and theIRES is less than five kilobases. As an alternative to an IRES, the DNAmay be separated by sequences encoding a 2A peptide, which self-cleavesin a post-translational event. See, e.g., M. L. Donnelly, et al, J. Gen.Virol., 78(Pt 1):13-21 (January 1997); Furler, S., et al, Gene Ther.,8(11):864-873 (June 2001); Klump H., et al., Gene Ther., 8(10):811-817(May 2001). This 2A peptide is significantly smaller than an IRES,making it well suited for use when space is a limiting factor. Moreoften, when the transgene is large, consists of multi-subunits, or twotransgenes are co-delivered, rAAV carrying the desired transgene(s) orsubunits are co-administered to allow them to concatamerize in vivo toform a single vector genome. In such an embodiment, a first AAV maycarry an expression cassette which expresses a single transgene and asecond AAV may carry an expression cassette which expresses a differenttransgene for co-expression in the host cell. However, the selectedtransgene may encode any biologically active product or other product,e.g., a product desirable for study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis invention.

2. Regulatory Elements

In addition to the major elements identified above for the minigene, thevector also includes conventional control elements which are operablylinked to the transgene in a manner which permits its transcription,translation and/or expression in a cell transfected with the plasmidvector or infected with the virus produced by the invention. As usedherein, “operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1promoter [Invitrogen]. Inducible promoters allow regulation of geneexpression and can be regulated by exogenously supplied compounds,environmental factors such as temperature, or the presence of a specificphysiological state, e.g., acute phase, a particular differentiationstate of the cell, or in replicating cells only. Inducible promoters andinducible systems are available from a variety of commercial sources,including, without limitation, Invitrogen, Clontech and Ariad. Manyother systems have been described and can be readily selected by one ofskill in the art. Examples of inducible promoters regulated byexogenously supplied compounds, include, the zinc-inducible sheepmetallothionine (MT) promoter, the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter, the T7 polymerase promoter system[International Patent Publication No. WO 98/10088]; the ecdysone insectpromoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)],the tetracycline-repressible system [Gossen et al, Proc. Natl. Acad.Sci. USA, 89:5547-5551 (1992)], the tetracycline-inducible system[Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al,Curr. Opin. Chem. Biol., 2:512-518 (1998)], the RU486-inducible system[Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, GeneTher., 4:432-441 (1997)] and the rapamycin-inducible system [Magari etal, J. Clin. Invest., 100:2865-2872 (1997)]. Other types of induciblepromoters which may be useful in this context are those which areregulated by a specific physiological state, e.g., temperature, acutephase, a particular differentiation state of the cell, or in replicatingcells only.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a gene operably linked to atissue-specific promoter. For instance, if expression in skeletal muscleis desired, a promoter active in muscle should be used. These includethe promoters from genes encoding skeletal β-actin, myosin light chain2A, dystrophin, muscle creatine kinase, as well as synthetic musclepromoters with activities higher than naturally-occurring promoters (seeLi et al., Nat. Biotech., 17:241-245 (1999)). Examples of promoters thatare tissue-specific are known for liver (albumin, Miyatake et al., J.Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig etal., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot etal., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et al.,Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein (Chen et al., J.Bone Miner. Res., 11:654-64 (1996)), lymphocytes (CD2, Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptorchain), neuronal such as neuron-specific enolase (NSE) promoter(Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)),neurofilament light-chain gene (Piccioli et al., Proc. Natl. Acad. Sci.USA, 88:5611-5 (1991)), and the neuron-specific vgf gene (Piccioli etal., Neuron, 15:373-84 (1995)), among others.

Optionally, plasmids carrying therapeutically useful transgenes may alsoinclude selectable markers or reporter genes may include sequencesencoding geneticin, hygromicin or purimycin resistance, among others.Such selectable reporters or marker genes (preferably located outsidethe viral genome to be rescued by the method of the invention) can beused to signal the presence of the plasmids in bacterial cells, such asampicillin resistance. Other components of the plasmid may include anorigin of replication. Selection of these and other promoters and vectorelements are conventional and many such sequences are available [see,e.g., Sambrook et al, and references cited therein].

The combination of the transgene, promoter/enhancer, and 5′ and 3′ AAVITRs is referred to as a “minigene” for ease of reference herein.Provided with the teachings of this invention, the design of such aminigene can be made by resort to conventional techniques.

3. Delivery of the Minigene to a Packaging Host Cell

The minigene can be carried on any suitable vector, e.g., a plasmid,which is delivered to a host cell. The plasmids useful in this inventionmay be engineered such that they are suitable for replication and,optionally, integration in prokaryotic cells, mammalian cells, or both.These plasmids (or other vectors carrying the 5′ AAV ITR-heterologousmolecule-3′ AAV ITR) contain sequences permitting replication of theminigene in eukaryotes and/or prokaryotes and selection markers forthese systems. Selectable markers or reporter genes may includesequences encoding geneticin, hygromicin or purimycin resistance, amongothers. The plasmids may also contain certain selectable reporters ormarker genes that can be used to signal the presence of the vector inbacterial cells, such as ampicillin resistance. Other components of theplasmid may include an origin of replication and an amplicon, such asthe amplicon system employing the Epstein Barr virus nuclear antigen.This amplicon system, or other similar amplicon components permit highcopy episomal replication in the cells. Preferably, the moleculecarrying the minigene is transfected into the cell, where it may existtransiently. Alternatively, the minigene (carrying the 5′ AAVITR-heterologous molecule-3′ ITR) may be stably integrated into thegenome of the host cell, either chromosomally or as an episome. Incertain embodiments, the minigene may be present in multiple copies,optionally in head-to-head, head-to-tail, or tail-to-tail concatamers.Suitable transfection techniques are known and may readily be utilizedto deliver the minigene to the host cell.

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, about 10 μg to about 50 μg DNA to about 1×10⁴ cells toabout 1×10¹³ cells, or about 1×10⁵ cells. However, the relative amountsof vector DNA to host cells may be adjusted, taking into considerationsuch factors as the selected vector, the delivery method and the hostcells selected.

B. Rep and Cap Sequences

In addition to the minigene, the host cell contains the sequences whichdrive expression of a novel AAV capsid protein of the invention (or acapsid protein comprising a fragment thereof) in the host cell and repsequences of the same source as the source of the AAV ITRs found in theminigene, or a cross-complementing source. The AAV cap and rep sequencesmay be independently obtained from an AAV source as described above andmay be introduced into the host cell in any manner known to one in theart as described above. Additionally, when pseudotyping an AAV vector in(e.g., an AAV9/HU.14 capsid), the sequences encoding each of theessential rep proteins may be supplied by different AAV sources (e.g.,AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8). For example, therep78/68 sequences may be from AAV2, whereas the rep52/40 sequences maybe from AAV8.

In one embodiment, the host cell stably contains the capsid proteinunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the capsid protein is expressedunder the control of an inducible promoter. In another embodiment, thecapsid protein is supplied to the host cell in trans. When delivered tothe host cell in trans, the capsid protein may be delivered via aplasmid which contains the sequences necessary to direct expression ofthe selected capsid protein in the host cell. Most desirably, whendelivered to the host cell in trans, the plasmid carrying the capsidprotein also carries other sequences required for packaging the rAAV,e.g., the rep sequences.

In another embodiment, the host cell stably contains the rep sequencesunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the essential rep proteins areexpressed under the control of an inducible promoter. In anotherembodiment, the rep proteins are supplied to the host cell in trans.When delivered to the host cell in trans, the rep proteins may bedelivered via a plasmid which contains the sequences necessary to directexpression of the selected rep proteins in the host cell. Mostdesirably, when delivered to the host cell in trans, the plasmidcarrying the capsid protein also carries other sequences required forpackaging the rAAV, e.g., the rep and cap sequences.

Thus, in one embodiment, the rep and cap sequences may be transfectedinto the host cell on a single nucleic acid molecule and exist stably inthe cell as an episome. In another embodiment, the rep and cap sequencesare stably integrated into the chromosome of the cell. Anotherembodiment has the rep and cap sequences transiently expressed in thehost cell. For example, a useful nucleic acid molecule for suchtransfection comprises, from 5′ to 3′, a promoter, an optional spacerinterposed between the promoter and the start site of the rep genesequence, an AAV rep gene sequence, and an AAV cap gene sequence.

Optionally, the rep and/or cap sequences may be supplied on a vectorthat contains other DNA sequences that are to be introduced into thehost cells. For instance, the vector may contain the rAAV constructcomprising the minigene. The vector may comprise one or more of thegenes encoding the helper functions, e.g., the adenoviral proteins E1,E2a, and E4 ORF6, and the gene for VAI RNA.

Preferably, the promoter used in this construct may be any of theconstitutive, inducible or native promoters known to one of skill in theart or as discussed above. In one embodiment, an AAV P5 promotersequence is employed. The selection of the AAV to provide any of thesesequences does not limit the invention.

In another preferred embodiment, the promoter for rep is an induciblepromoter, such as are discussed above in connection with the transgeneregulatory elements. One preferred promoter for rep expression is the T7promoter. The vector comprising the rep gene regulated by the T7promoter and the cap gene, is transfected or transformed into a cellwhich either constitutively or inducibly expresses the T7 polymerase.See International Patent Publication No. WO 98/10088, published Mar. 12,1998.

The spacer is an optional element in the design of the vector. Thespacer is a DNA sequence interposed between the promoter and the repgene ATG start site. The spacer may have any desired design; that is, itmay be a random sequence of nucleotides, or alternatively, it may encodea gene product, such as a marker gene. The spacer may contain geneswhich typically incorporate start/stop and polyA sites. The spacer maybe a non-coding DNA sequence from a prokaryote or eukaryote, arepetitive non-coding sequence, a coding sequence withouttranscriptional controls or a coding sequence with transcriptionalcontrols. Two exemplary sources of spacer sequences are the phage laddersequences or yeast ladder sequences, which are available commercially,e.g., from Gibco or Invitrogen, among others. The spacer may be of anysize sufficient to reduce expression of the rep78 and rep68 geneproducts, leaving the rep52, rep40 and cap gene products expressed atnormal levels. The length of the spacer may therefore range from about10 by to about 10.0 kbp, preferably in the range of about 100 bp toabout 8.0 kbp. To reduce the possibility of recombination, the spacer ispreferably less than 2 kbp in length; however, the invention is not solimited.

Although the molecule(s) providing rep and cap may exist in the hostcell transiently (i.e., through transfection), it is preferred that oneor both of the rep and cap proteins and the promoter(s) controllingtheir expression be stably expressed in the host cell, e.g., as anepisome or by integration into the chromosome of the host cell. Themethods employed for constructing embodiments of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above. While thisspecification provides illustrative examples of specific constructs,using the information provided herein, one of skill in the art mayselect and design other suitable constructs, using a choice of spacers,P5 promoters, and other elements, including at least one translationalstart and stop signal, and the optional addition of polyadenylationsites.

In another embodiment of this invention, the rep or cap protein may beprovided stably by a host cell.

C. The Helper Functions

The packaging host cell also requires helper functions in order topackage the rAAV of the invention. Optionally, these functions may besupplied by a herpesvirus. Most desirably, the necessary helperfunctions are each provided from a human or non-human primate adenovirussource, such as those described above and/or are available from avariety of sources, including the American Type Culture Collection(ATCC), Manassas, Va. (US). In one currently preferred embodiment, thehost cell is provided with and/or contains an E1a gene product, an E1bgene product, an E2a gene product, and/or an E4 ORF6 gene product. Thehost cell may contain other adenoviral genes such as VAI RNA, but thesegenes are not required. In a preferred embodiment, no other adenovirusgenes or gene functions are present in the host cell.

By “adenoviral DNA which expresses the E1a gene product”, it is meantany adenovirus sequence encoding E1a or any functional E1a portion.Adenoviral DNA which expresses the E2a gene product and adenoviral DNAwhich expresses the E4 ORF6 gene products are defined similarly. Alsoincluded are any alleles or other modifications of the adenoviral geneor functional portion thereof. Such modifications may be deliberatelyintroduced by resort to conventional genetic engineering or mutagenictechniques to enhance the adenoviral function in some manner, as well asnaturally occurring allelic variants thereof. Such modifications andmethods for manipulating DNA to achieve these adenovirus gene functionsare known to those of skill in the art.

The adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products, as well asany other desired helper functions, can be provided using any means thatallows their expression in a cell. Each of the sequences encoding theseproducts may be on a separate vector, or one or more genes may be on thesame vector. The vector may be any vector known in the art or disclosedabove, including plasmids, cosmids and viruses. Introduction into thehost cell of the vector may be achieved by any means known in the art oras disclosed above, including transfection, infection, electroporation,liposome delivery, membrane fusion techniques, high velocity DNA-coatedpellets, viral infection and protoplast fusion, among others. One ormore of the adenoviral genes may be stably integrated into the genome ofthe host cell, stably expressed as episomes, or expressed transiently.The gene products may all be expressed transiently, on an episome orstably integrated, or some of the gene products may be expressed stablywhile others are expressed transiently. Furthermore, the promoters foreach of the adenoviral genes may be selected independently from aconstitutive promoter, an inducible promoter or a native adenoviralpromoter. The promoters may be regulated by a specific physiologicalstate of the organism or cell (i.e., by the differentiation state or inreplicating or quiescent cells) or by exogenously added factors, forexample.

D. Host Cells and Packaging Cell Lines

The host cell itself may be selected from any biological organism,including prokaryotic (e.g., bacterial) cells, and eukaryotic cells,including, insect cells, yeast cells and mammalian cells. Particularlydesirable host cells are selected from among any mammalian species,including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2,BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293cells (which express functional adenoviral E1), Saos, C2C12, L cells,HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cellsderived from mammals including human, monkey, mouse, rat, rabbit, andhamster. The selection of the mammalian species providing the cells isnot a limitation of this invention; nor is the type of mammalian cell,i.e., fibroblast, hepatocyte, tumor cell, etc. The requirements for thecell used is that it not carry any adenovirus gene other than E1, E2aand/or E4 ORF6; it not contain any other virus gene which could resultin homologous recombination of a contaminating virus during theproduction of rAAV; and it is capable of infection or transfection ofDNA and expression of the transfected DNA. In a preferred embodiment,the host cell is one that has rep and cap stably transfected in thecell.

One host cell useful in the present invention is a host cell stablytransformed with the sequences encoding rep and cap, and which istransfected with the adenovirus E1, E2a, and E4ORF6 DNA and a constructcarrying the minigene as described above. Stable rep and/or capexpressing cell lines, such as B-50 (International Patent ApplicationPublication No. WO 99/15685), or those described in U.S. Pat. No.5,658,785, may also be similarly employed. Another desirable host cellcontains the minimum adenoviral DNA which is sufficient to express E4ORF6. Yet other cell lines can be constructed using the novel AAV9 capsequences of the invention.

The preparation of a host cell according to this invention involvestechniques such as assembly of selected DNA sequences. This assembly maybe accomplished utilizing conventional techniques. Such techniquesinclude cDNA and genomic cloning, which are well known and are describedin Sambrook et al., cited above, use of overlapping oligonucleotidesequences of the adenovirus and AAV genomes, combined with polymerasechain reaction, synthetic methods, and any other suitable methods whichprovide the desired nucleotide sequence.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation, and/or infection by hybridadenovirus/AAV vectors into cell lines such as the human embryonickidney cell line HEK 293 (a human kidney cell line containing functionaladenovirus E1 genes which provides trans-acting E1 proteins).

The AAV9/HU.14 based vectors which are generated by one of skill in theart are beneficial for gene delivery to selected host cells and genetherapy patients since no neutralization antibodies to AAV9/HU.14 havebeen found in the human population. One of skill in the art may readilyprepare other rAAV viral vectors containing the AAV9/HU.14 capsidproteins provided herein using a variety of techniques known to those ofskill in the art. One may similarly prepare still other rAAV viralvectors containing AAV9/HU.14 sequence and AAV capsids from anothersource.

One of skill in the art will readily understand that the novel AAVsequences of the invention can be readily adapted for use in these andother viral vector systems for in vitro, ex vivo or in vivo genedelivery. Similarly, one of skill in the art can readily select otherfragments of the AAV genome of the invention for use in a variety ofrAAV and non-rAAV vector systems. Such vectors systems may include,e.g., lentiviruses, retroviruses, poxviruses, vaccinia viruses, andadenoviral systems, among others. Selection of these vector systems isnot a limitation of the present invention.

Thus, the invention further provides vectors generated using the nucleicacid and amino acid sequences of the novel AAV of the invention. Suchvectors are useful for a variety of purposes, including for delivery oftherapeutic molecules and for use in vaccine regimens. Particularlydesirable for delivery of therapeutic molecules are recombinant AAVcontaining capsids of the novel AAV of the invention. These, or othervector constructs containing novel AAV sequences of the invention may beused in vaccine regimens, e.g., for co-delivery of a cytokine, or fordelivery of the immunogen itself.

IV. Recombinant Viruses and Uses Therefor

Using the techniques described herein, one of skill in the art cangenerate a rAAV having a capsid of an AAV of the invention or having acapsid containing one or more fragments of an AAV of the invention. Inone embodiment, a full-length capsid from a single AAV, e.g., hu.14/AAV9[SEQ ID NO: 123] can be utilized. In another embodiment, a full-lengthcapsid may be generated which contains one or more fragments of thenovel AAV capsid of the invention fused in frame with sequences fromanother selected AAV, or from heterologous (i.e., non-contiguous)portions of the same AAV. For example, a rAAV may contain one or more ofthe novel hypervariable region sequences of AAV9/HU.14. Alternatively,the unique AAV sequences of the invention may be used in constructscontaining other viral or non-viral sequences. Optionally, a recombinantvirus may carry AAV rep sequences encoding one or more of the AAV repproteins.

A. Delivery of Viruses

In another aspect, the present invention provides a method for deliveryof a transgene to a host which involves transfecting or infecting aselected host cell with a recombinant viral vector generated with theAAV9/HU.14 sequences (or functional fragments thereof) of the invention.Methods for delivery are well known to those of skill in the art and arenot a limitation of the present invention.

In one desirable embodiment, the invention provides a method forAAV-mediated delivery of a transgene to a host. This method involvestransfecting or infecting a selected host cell with a recombinant viralvector containing a selected transgene under the control of sequencesthat direct expression thereof and AAV9 capsid proteins.

Optionally, a sample from the host may be first assayed for the presenceof antibodies to a selected AAV source (e.g., a serotype). A variety ofassay formats for detecting neutralizing antibodies are well known tothose of skill in the art. The selection of such an assay is not alimitation of the present invention. See, e.g., Fisher et al, NatureMed., 3(3):306-312 (March 1997) and W. C. Manning et al, Human GeneTherapy, 9:477-485 (Mar. 1, 1998). The results of this assay may be usedto determine which AAV vector containing capsid proteins of a particularsource are preferred for delivery, e.g., by the absence of neutralizingantibodies specific for that capsid source.

In one aspect of this method, the delivery of vector with AAV capsidproteins of the invention may precede or follow delivery of a gene via avector with a different AAV capsid protein. Thus, gene delivery via rAAVvectors may be used for repeat gene delivery to a selected host cell.Desirably, subsequently administered rAAV vectors carry the sametransgene as the first rAAV vector, but the subsequently administeredvectors contain capsid proteins of sources (and preferably, differentserotypes) which differ from the first vector. For example, if a firstvector has AAV9/HU.14 capsid proteins, subsequently administered vectorsmay have capsid proteins selected from among the other AAV, optionally,from another serotype or from another clade.

Optionally, multiple rAAV vectors can be used to deliver largetransgenes or multiple transgenes by co-administration of rAAV vectorsconcatamerize in vivo to form a single vector genome. In such anembodiment, a first AAV may carry an expression cassette which expressesa single transgene (or a subunit thereof) and a second AAV may carry anexpression cassette which expresses a second transgene (or a differentsubunit) for co-expression in the host cell. A first AAV may carry anexpression cassette which is a first piece of a polycistronic construct(e.g., a promoter and transgene, or subunit) and a second AAV may carryan expression cassette which is a second piece of a polycistronicconstruct (e.g., transgene or subunit and a polyA sequence). These twopieces of a polycistronic construct concatamerize in vivo to form asingle vector genome that co-expresses the transgenes delivered by thefirst and second AAV. In such embodiments, the rAAV vector carrying thefirst expression cassette and the rAAV vector carrying the secondexpression cassette can be delivered in a single pharmaceuticalcomposition. In other embodiments, the two or more rAAV vectors aredelivered as separate pharmaceutical compositions which can beadministered substantially simultaneously, or shortly before or afterone another.

The above-described recombinant vectors may be delivered to host cellsaccording to published methods. The rAAV, preferably suspended in aphysiologically compatible carrier, may be administered to a human ornon-human mammalian patient. Suitable carriers may be readily selectedby one of skill in the art in view of the indication for which thetransfer virus is directed. For example, one suitable carrier includessaline, which may be formulated with a variety of buffering solutions(e.g., phosphate buffered saline). Other exemplary carriers includesterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran,agar, pectin, peanut oil, sesame oil, and water. The selection of thecarrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The vectors are administered in sufficient amounts to transfect thecells and to provide sufficient levels of gene transfer and expressionto provide a therapeutic benefit without undue adverse effects, or withmedically acceptable physiological effects, which can be determined bythose skilled in the medical arts. Conventional and pharmaceuticallyacceptable routes of administration include, but are not limited to,direct delivery to a desired organ (e.g., the liver (optionally via thehepatic artery) or lung), oral, inhalation, intranasal, intratracheal,intraarterial, intraocular, intravenous, intramuscular, subcutaneous,intradermal, and other parental routes of administration. Routes ofadministration may be combined, if desired.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectivehuman dosage of the viral vector is generally in the range of from about0.1 mL to about 100 mL of solution containing concentrations of fromabout 1×10⁹ to 1×10¹⁶ genomes virus vector. A preferred human dosage fordelivery to large organs (e.g., liver, muscle, heart and lung) may beabout 5×10¹⁰ to 5×10¹³ AAV genomes per 1 kg, at a volume of about 1 to100 mL. A preferred dosage for delivery to eye is about 5×10⁹ to 5×10¹²genome copies, at a volume of about 0.1 mL to 1 mL. The dosage will beadjusted to balance the therapeutic benefit against any side effects andsuch dosages may vary depending upon the therapeutic application forwhich the recombinant vector is employed. The levels of expression ofthe transgene can be monitored to determine the frequency of dosageresulting in viral vectors, preferably AAV vectors containing theminigene. Optionally, dosage regimens similar to those described fortherapeutic purposes may be utilized for immunization using thecompositions of the invention.

Examples of therapeutic products and immunogenic products for deliveryby the AAV-containing vectors of the invention are provided below. Thesevectors may be used for a variety of therapeutic or vaccinal regimens,as described herein. Additionally, these vectors may be delivered incombination with one or more other vectors or active ingredients in adesired therapeutic and/or vaccinal regimen.

B. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), platelet-derived growth factor (PDGF), insulin growth factors Iand II (IGF-I and IGF-II), any one of the transforming growth factor αsuperfamily, including TGFα, activins, inhibins, or any of the bonemorphogenic proteins (BMP) BMPs 1-15, any one of theheregluin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors α and β,interferons α, β, and γ, stem cell factor, flk-2/flt3 ligand. Geneproducts produced by the immune system are also useful in the invention.These include, without limitations, immunoglobulins IgG, IgM, IgA, IgDand IgE, chimeric immunoglobulins, humanized antibodies, single chainantibodies, T cell receptors, chimeric T cell receptors, single chain Tcell receptors, class I and class II MHC molecules, as well asengineered immunoglobulins and MHC molecules. Useful gene products alsoinclude complement regulatory proteins such as complement regulatoryproteins, membrane cofactor protein (MCP), decay accelerating factor(DAF), CR1, CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation and/or lipid modulation, including the lowdensity lipoprotein (LDL) receptor, high density lipoprotein (HDL)receptor, the very low density lipoprotein (VLDL) receptor, andscavenger receptors. The invention also encompasses gene products suchas members of the steroid hormone receptor superfamily includingglucocorticoid receptors and estrogen receptors, Vitamin D receptors andother nuclear receptors. In addition, useful gene products includetranscription factors such as jun, fos, max, mad, serum response factor(SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1,CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilmstumor protein, ETS-binding protein, STAT, GATA-box binding proteins,e.g., GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin gene product [e.g., a mini- or micro-dystrophin]. Still otheruseful gene products include enzymes such as may be useful in enzymereplacement therapy, which is useful in a variety of conditionsresulting from deficient activity of enzyme. For example, enzymes thatcontain mannose-6-phosphate may be utilized in therapies for lysosomalstorage diseases (e.g., a suitable gene includes that encodingβ-glucuronidase (GUSB)).

Still other useful gene products include those used for treatment ofhemophilia, including hemophilia B (including Factor IX) and hemophiliaA (including Factor VIII and its variants, such as the light chain andheavy chain of the heterodimer and the B-deleted domain; U.S. Pat. No.6,200,560 and U.S. Pat. No. 6,221,349). The Factor VIII gene codes for2351 amino acids and the protein has six domains, designated from theamino to the terminal carboxy terminus as A1-A2-B-A3-C1-C2 [Wood et al,Nature, 312:330 (1984); Vehar et al., Nature 312:337 (1984); and Tooleet al, Nature, 342:337 (1984)]. Human Factor VIII is processed withinthe cell to yield a heterodimer primarily comprising a heavy chaincontaining the A1, A2 and B domains and a light chain containing the A3,C1 and C2 domains. Both the single chain polypeptide and the heterodimercirculate in the plasma as inactive precursors, until activated bythrombin cleavage between the A2 and B domains, which releases the Bdomain and results in a heavy chain consisting of the A1 and A2 domains.The B domain is deleted in the activated procoagulant form of theprotein. Additionally, in the native protein, two polypeptide chains(“a” and “b”), flanking the B domain, are bound to a divalent calciumcation.

In some embodiments, the minigene comprises first 57 base pairs of theFactor VIII heavy chain which encodes the 10 amino acid signal sequence,as well as the human growth hormone (hGH) polyadenylation sequence. Inalternative embodiments, the minigene further comprises the A1 and A2domains, as well as 5 amino acids from the N-terminus of the B domain,and/or 85 amino acids of the C-terminus of the B domain, as well as theA3, C1 and C2 domains. In yet other embodiments, the nucleic acidsencoding Factor VIII heavy chain and light chain are provided in asingle minigene separated by 42 nucleic acids coding for 14 amino acidsof the B domain [U.S. Pat. No. 6,200,560].

As used herein, a therapeutically effective amount is an amount of AAVvector that produces sufficient amounts of Factor VIII to decrease thetime it takes for a subject's blood to clot. Generally, severehemophiliacs having less than 1% of normal levels of Factor VIII have awhole blood clotting time of greater than 60 minutes as compared toapproximately 10 minutes for non-hemophiliacs.

The present invention is not limited to any specific Factor VIIIsequence. Many natural and recombinant forms of Factor VIII have beenisolated and generated. Examples of naturally occurring and recombinantforms of Factor VII can be found in the patent and scientific literatureincluding, U.S. Pat. No. 5,563,045, U.S. Pat. No. 5,451,521, U.S. Pat.No. 5,422,260, U.S. Pat. No. 5,004,803, U.S. Pat. No. 4,757,006, U.S.Pat. No. 5,661,008, U.S. Pat. No. 5,789,203, U.S. Pat. No. 5,681,746,U.S. Pat. No. 5,595,886, U.S. Pat. No. 5,045,455, U.S. Pat. No.5,668,108, U.S. Pat. No. 5,633,150, U.S. Pat. No. 5,693,499, U.S. Pat.No. 5,587,310, U.S. Pat. No. 5,171,844, U.S. Pat. No. 5,149,637, U.S.Pat. No. 5,112,950, U.S. Pat. No. 4,886,876; International PatentPublication Nos. WO 94/11503, WO 87/07144, WO 92/16557, WO 91/09122, WO97/03195, WO 96/21035, and WO 91/07490; European Patent Application Nos.EP 0 672 138, EP 0 270 618, EP 0 182 448, EP 0 162 067, EP 0 786 474, EP0 533 862, EP 0 506 757, EP 0 874 057,EP 0 795 021, EP 0 670 332, EP 0500 734, EP 0 232 112, and EP 0 160 457; Sanberg et al., XXth Int.Congress of the World Fed. Of Hemophilia (1992), and Lind et al., Eur.J. Biochem., 232:19 (1995).

Nucleic acids sequences coding for the above-described Factor VIII canbe obtained using recombinant methods or by deriving the sequence from avector known to include the same. Furthermore, the desired sequence canbe isolated directly from cells and tissues containing the same, usingstandard techniques, such as phenol extraction and PCR of cDNA orgenomic DNA [See, e.g., Sambrook et al]. Nucleotide sequences can alsobe produced synthetically, rather than cloned. The complete sequence canbe assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence [See, e.g., Edge,Nature 292:757 (1981); Nambari et al, Science, 223:1299 (1984); and Jayet al, J. Biol. Chem. 259:6311 (1984).

Furthermore, the invention is not limited to human Factor VIII. Indeed,it is intended that the present invention encompass Factor VIII fromanimals other than humans, including but not limited to companionanimals (e.g., canine, felines, and equines), livestock (e.g., bovines,caprines and ovines), laboratory animals, marine mammals, large cats,etc.

The AAV vectors may contain a nucleic acid coding for fragments ofFactor VIII which is itself not biologically active, yet whenadministered into the subject improves or restores the blood clottingtime. For example, as discussed above, the Factor VIII protein comprisestwo polypeptide chains: a heavy chain and a light chain separated by aB-domain which is cleaved during processing. As demonstrated by thepresent invention, co-tranducing recipient cells with the Factor VIIIheavy and light chains leads to the expression of biologically activeFactor VIII. Because most hemophiliacs contain a mutation or deletion inonly one of the chains (e.g., heavy or light chain), it may be possibleto administer only the chain defective in the patient to supply theother chain.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene is particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce “self”-directed antibodies. Tcell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors (TCRs) that bind to endogenousantigens and initiate the inflammatory cascade associated withautoimmune diseases.

C. Immunogenic Transgenes

Suitably, the AAV vectors of the invention avoid the generation ofimmune responses to the AAV sequences contained within the vector.However, these vectors may nonetheless be formulated in a manner thatpermits the expression of a transgene carried by the vectors to inducean immune response to a selected antigen. For example, in order topromote an immune response, the transgene may be expressed from aconstitutive promoter, the vector can be adjuvanted as described herein,and/or the vector can be put into degenerating tissue.

Examples of suitable immunogenic transgenes include those selected froma variety of viral families. Examples of desirable viral familiesagainst which an immune response would be desirable include, thepicornavirus family, which includes the genera rhinoviruses, which areresponsible for about 50% of cases of the common cold; the generaenteroviruses, which include polioviruses, coxsackieviruses,echoviruses, and human enteroviruses such as hepatitis A virus; and thegenera apthoviruses, which are responsible for foot and mouth diseases,primarily in non-human animals. Within the picornavirus family ofviruses, target antigens include the VP1, VP2, VP3, VP4, and VPG. Otherviral families include the astroviruses and the calcivirus family. Thecalcivirus family encompasses the Norwalk group of viruses, which are animportant causative agent of epidemic gastroenteritis. Still anotherviral family desirable for use in targeting antigens for inducing immuneresponses in humans and non-human animals is the togavirus family, whichincludes the genera alphavirus, which include Sindbis viruses, RossRivervirus, and Venezuelan, Eastern & Western Equine encephalitis, andrubivirus, including Rubella virus. The flaviviridae family includesdengue, yellow fever, Japanese encephalitis, St. Louis encephalitis andtick borne encephalitis viruses. Other target antigens may be generatedfrom the Hepatitis C or the coronavirus family, which includes a numberof non-human viruses such as infectious bronchitis virus (poultry),porcine transmissible gastroenteric virus (pig), porcine hemagglutinatinencephalomyelitis virus (pig), feline infectious peritonitis virus(cat), feline enteric coronavirus (cat), canine coronavirus (dog), andhuman respiratory coronaviruses, which may cause the common cold and/ornon-A, B or C hepatitis, and which include the putative cause of suddenacute respiratory syndrome (SARS). Within the coronavirus family, targetantigens include the E1 (also called M or matrix protein), E2 (alsocalled S or Spike protein), E3 (also called HE or hemagglutin-elterose)glycoprotein (not present in all coronaviruses), or N (nucleocapsid).Still other antigens may be targeted against the arterivirus family andthe rhabdovirus family. The rhabdovirus family includes the generavesiculovirus (e.g., Vesicular Stomatitis Virus), and the generallyssavirus (e.g., rabies). Within the rhabdovirus family, suitableantigens may be derived from the G protein or the N protein. The familyfiloviridae, which includes hemorrhagic fever viruses such as Marburgand Ebola virus may be a suitable source of antigens. The paramyxovirusfamily includes parainfluenza Virus Type 1, parainfluenza Virus Type 3,bovine parainfluenza Virus Type 3, rubulavirus (mumps virus,parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastledisease virus (chickens), rinderpest, morbillivirus, which includesmeasles and canine distemper, and pneumovirus, which includesrespiratory syncytial virus. The influenza virus is classified withinthe family orthomyxovirus and is a suitable source of antigen (e.g., theHA protein, the N1 protein). The bunyavirus family includes the generabunyavirus (California encephalitis, La Crosse), phlebovirus (RiftValley Fever), hantavirus (puremala is a hemahagin fever virus),nairovirus (Nairobi sheep disease) and various unassigned bungaviruses.The arenavirus family provides a source of antigens against LCM andLassa fever virus. Another source of antigens is the bornavirus family.The reovirus family includes the genera reovirus, rotavirus (whichcauses acute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue). The retrovirus family includes the sub-family oncorivirinalwhich encompasses such human and veterinary diseases as feline leukemiavirus, HTLVI and HTLVII, lentivirinal (which includes HIV, simianimmunodeficiency virus, feline immunodeficiency virus, equine infectiousanemia virus, and spumavirinal). The papovavirus family includes thesub-family polyomaviruses (BKU and JCU viruses) and the sub-familypapillomavirus (associated with cancers or malignant progression ofpapilloma). The adenovirus family includes viruses (EX, AD7, ARD, O.B.)which cause respiratory disease and/or enteritis. The parvovirus familyincludes feline parvovirus (feline enteritis), felinepanleucopeniavirus, canine parvovirus, and porcine parvovirus. Theherpesvirus family includes the sub-family alphaherpesvirinae, whichencompasses the genera simplexvirus (HSVI, HSVII), varicellovirus(pseudorabies, varicella zoster) and the sub-family betaherpesvirinae,which includes the genera cytomegalovirus (HCMV, muromegalovirus) andthe sub-family gammaherpesvirinae, which includes the generalymphocryptovirus, EBV (Burkitts lymphoma), human herpesviruses 6A, 6Band 7, Kaposi's sarcoma-associated herpesvirus and cercopithecineherpesvirus (B virus), infectious rhinotracheitis, Marek's diseasevirus, and rhadinovirus. The poxvirus family includes the sub-familychordopoxvirinae, which encompasses the genera orthopoxvirus (Variolamajor (Smallpox) and Vaccinia (Cowpox)), parapoxvirus, avipoxvirus,capripoxvirus, leporipoxvirus, suipoxvirus, and the sub-familyentomopoxvirinae. The hepadnavirus family includes the Hepatitis Bvirus. One unclassified virus which may be suitable source of antigensis the Hepatitis delta virus, Hepatitis E virus, and prions. Anothervirus which is a source of antigens is Nipan Virus. Still other viralsources may include avian infectious bursal disease virus and porcinerespiratory and reproductive syndrome virus. The alphavirus familyincludes equine arteritis virus and various Encephalitis viruses.

The present invention may also encompass immunogens which are useful toimmunize a human or non-human animal against other pathogens includingbacteria, fungi, parasitic microorganisms or multicellular parasiteswhich infect human and non-human vertebrates, or from a cancer cell ortumor cell. Examples of bacterial pathogens include pathogenicgram-positive cocci include pneumococci; staphylococci (and the toxinsproduced thereby, e.g., enterotoxin B); and streptococci. Pathogenicgram-negative cocci include meningococcus; gonococcus. Pathogenicenteric gram-negative bacilli include enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (which causes chancroid); brucellaspecies (brucellosis); Francisella tularensis (which causes tularemia);Yersinia pestis (plague) and other yersinia (pasteurella);streptobacillus moniliformis and spirillum; Gram-positive bacilliinclude listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracia(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism (Clostridum botulinum and its toxin); Clostridium perfringensand its epsilon toxin; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude glanders (Burkholderia mallei); actinomycosis; nocardiosis;cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis;candidiasis, aspergillosis, and mucormycosis; sporotrichosis;paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma andchromomycosis; and dermatophytosis. Rickettsial infections includeTyphus fever, Rocky Mountain spotted fever, Q fever (Coxiella burnetti),and Rickettsialpox. Examples of mycoplasma and chlamydial infectionsinclude: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis;and perinatal chlamydial infections. Pathogenic eukaryotes encompasspathogenic protozoans and helminths and infections produced therebyinclude: amebiasis; malaria; leishmaniasis; trypanosomiasis;toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii;babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis;nematodes; trematodes or flukes; and cestode (tapeworm) infections.

Many of these organisms and/or the toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracia (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the vectors of the invention to deliver immunogensagainst the variable region of the T cells elicit an immune responseincluding CTLs to eliminate those T cells. In rheumatoid arthritis (RA),several specific variable regions of TCRs which are involved in thedisease have been characterized. These TCRs include V-3, V-14, V-17 andV-17. Thus, delivery of a nucleic acid sequence that encodes at leastone of these polypeptides will elicit an immune response that willtarget T cells involved in RA. In multiple sclerosis (MS), severalspecific variable regions of TCRs which are involved in the disease havebeen characterized. These TCRs include V-7 and V-10. Thus, delivery of anucleic acid sequence that encodes at least one of these polypeptideswill elicit an immune response that will target T cells involved in MS.In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V-6,V-8, V-14 and V-16, V-3C, V-7, V-14, V-15, V-16, V-28 and V-12. Thus,delivery of a nucleic acid molecule that encodes at least one of thesepolypeptides will elicit an immune response that will target T cellsinvolved in scleroderma.

Thus, a rAAV-derived recombinant viral vector of the invention providesan efficient gene transfer vehicle which can deliver a selectedtransgene to a selected host cell in vivo or ex vivo even where theorganism has neutralizing antibodies to one or more AAV sources. In oneembodiment, the rAAV and the cells are mixed ex vivo; the infected cellsare cultured using conventional methodologies; and the transduced cellsare re-infused into the patient.

These compositions are particularly well suited to gene delivery fortherapeutic purposes and for immunization, including inducing protectiveimmunity. Further, the compositions of the invention may also be usedfor production of a desired gene product in vitro. For in vitroproduction, a desired product (e.g., a protein) may be obtained from adesired culture following transfection of host cells with a rAAVcontaining the molecule encoding the desired product and culturing thecell culture under conditions which permit expression. The expressedproduct may then be purified and isolated, as desired. Suitabletechniques for transfection, cell culturing, purification, and isolationare known to those of skill in the art.

The following examples illustrate several aspects and embodiments of theinvention.

EXAMPLE 1 Computational Analysis of Primate AAV Sequences

A. Collection of Primate Tissues

Sources of nonhuman primate tissues were described previously [N.Muzyczka, K. I. Berns, in Fields Virology D. M. Knipe, P. M. Howley,Eds. (Lippincott Williams & Wilkins, Philadelphia, 2001), vol. 2, pp.2327-2359]. Human tissues were collected from either surgical proceduresor postmortem examination or organ donors through two major nationalhuman tissue providers, Cooperative Human Tissue Network (CHTN) andNational Disease Research Interchange (NDRI). Human tissues used forthis study were comprised of 18 different tissue types that includedcolon, liver, lung, spleen, kidney, brain, small bowel, bone marrow,heart, lymph nodes, skeletal muscle, ovary, pancreas, stomach,esophagus, cervix, testis and prostate. The tissue samples came from adiverse group of individuals of different gender, races (Caucasian,African-American, Asian and Hispanic) and ages (23-83 years). Among 259samples from 250 individuals analyzed, approximately 28% of tissues wereassociated with pathology.

B. Detection and Isolation of AAV Sequences

Total cellular DNAs were extracted from human and nonhuman primatetissues as described previously [R. W. Atchison, et al., Science 194,754-756 (1965)]. Molecular prevalence and tissue distribution of AAVs inhumans were determined by either signature or full-length cap PCR usingthe primers and conditions that were similar to those used for thenonhuman primate analysis. The same PCR cloning strategy used for theisolation and characterization of an expanded family of AAVs in nonhumanprimates was deployed in the isolation of AAVs from selected humantissues. Briefly, a 3.1 kb fragment containing a part of rep and fulllength cap sequence was amplified from tissue DNAs by PCR andTopo-cloned (Invitrogen). The human AAV clones were initially analyzedby restriction mapping to help identify diversity of AAV sequences,which were subsequently subjected to full sequence analysis by SeqWright(SeqWright, Houston, Tex.) with an accuracy of 99.9%. A total of 67capsid clones isolated from human tissues were characterized(hu.1-hu.67). From nonhuman primate tissues, 86 cap clones weresequenced, among which 70 clones were from rhesus macaques, 6 clonesfrom cynomologus macaques, 3 clones from pigtailed macaques, 2 clonesfrom a baboon and 5 clones from a chimpanzee.

C. Analysis of AAV Sequences

From all contiguous sequences, AAV capsid viral protein (vp1) openreading frames (ORFs) were analyzed. The AAV capsid VP1 proteinsequences were aligned with the ClustalX1.81™ program [H. D. Mayor, J.L. Melnick, Nature 210, 331-332 (1966)] and an in-frame DNA alignmentwas produced with the BioEdit™ [U. Bantel-Schaal, H. Zur Hausen,Virology 134, 52-63 (1984)] software package. Phylogenies were inferredwith the MEGA™ v2.1 and the TreePuzzle™ package. Neighbor-Joining,Maximum Parsimony, and Maximum Likelihood [M. Nei, S. Kumar, MolecularEvolution and Phylogenetics (Oxford University Press, New York, 2000);H. A. Schmidt, K. Strimmer, M. Vingron, A. von Haeseler, Bioinformatics18, 502-4 (March, 2002); N. Saitou, M. Nei, Mol Biol Evol 4, 406-25(July, 1987)] algorithms were used to confirm similar clustering ofsequences in monophylic groups.

Clades were then defined from a Neighbor-Joining phylogenetic tree ofall protein sequences. The amino-acid distances were estimated by makinguse of Poisson-correction. Bootstrap analysis was performed with a 1000replicates. Sequences were considered monophylic when they had aconnecting node within a 0.05 genetic distance. A group of sequencesoriginating from 3 or more sources was considered a clade. The phylogenyof AAV was further evaluated for evidence of recombination through asequential analysis. Homoplasy was screened for by implementation of theSplit Decomposition algorithm [H. J. Bandelt, A. W. Dress, MolPhylogenet Evol 1, 242-52 (September 1992)]. Splits that were picked upin this manner were then further analyzed for recombination making useof the Bootscan algorithm in the Simplot software [M. Nei and S. Kumar,Molecular Evolution and Phylogenetics (Oxford University Press, NewYork, 2000)]. A sliding window of 400 nt (10 nt/step) was used to obtain100 bootstrap replicate neighbor-joining trees. Subsequently, SplitDecomposition and Neighbor-Joining phylogenies were inferred from theputative recombination fragments. Significant improvement of bootstrapvalues, reduction of splits and regrouping of the hybrid sequences withtheir parental sources were considered the criterion for recombination.

A number of different cap sequences amplified from 8 different humansubjects showed phylogenetic relationships to AAV2 (5′) and AAV3 (3′)around a common breakpoint at position 1400 of the Cap DNA sequence,consistent with recombination and the formation of a hybrid virus. Thisis the general region of the cap gene where recombination was detectedfrom isolates from a mesenteric lymph node of a rhesus macaque [Gao etal., Proc Natl Acad Sci USA 100, 6081-6086 (May 13, 2002)]. An overallcodon based Z-test for selection was performed implementing theNeib-Gojobori method [R. M. Kotin, Hum Gene Ther 5, 793-801 (July,1994)].

The phylogenetic analyses were repeated excluding the clones that werepositively identified as hybrids. In this analysis, goose and avian AAVswere included as outgroups [(I. Bossis, J. A. Chiorini, J Virol 77,6799-810 (Jun. 2003)]. FIG. 1 is a neighbor-joining tree; similarrelationships were obtained using maximum parsimony and maximumlikelihood analyses.

This analysis demonstrated 11 phylogenetic groups, which are summarizedin Table 1. The species origin of the 6 AAV clades and 5 individual AAVclones (or sets of clones) is represented by the number or sources fromwhich the sequences were retrieved in the sampling. The total number ofsequences gathered per species and per grouping is shown in betweenbrackets. References for previously described sequences per clade are inthe right column. Rhesus—rhesus macaques; cyno—cynomologus macaques;chimp—chimpanzees; pigtail—pigtail macaques.

TABLE 1 Classification of the number of sources (sequences) per speciesand per clade or clone Human Rhesus Cyno Baboon Chimp Pigtail Clade/representative A/AAV1 (AAV6) 3(4) B/AAV2 12(22) C/AAV2-AAV3  8(17)hybrid D/AAV7 5(10) 5(5) E/AAV8 7(9) 7(16) 1(2) 1(3) F/AAV9 3(3) ClonesAAV3 AAV4 1(3)  AAV5 Ch.5 1(1) Rh.8 2(2) 

Since, as noted above, recombination is not implemented in the standardphylogenetic algorithms used, in order to build a proper phylogenetictree, those sequences were excluded from the analysis, of which theirrecombinative ancestry was established. A neighbor-joining analysis ofall non-recombined sequences is represented side by side with the cladesthat did evolve making use of recombination. A similar output wasgenerated with the different algorithm used and with the nucleotidesequence as input.

Additional experiments were performed to evaluate the relationship ofphylogenetic relatedness to function as measured by serologic activityand tropism, as described in the following examples.

EXAMPLE 2 Serological Analysis of Novel Human AAVs

The last clade obtained as described in the preceding example wasderived from isolates of 3 humans and did not contain a previouslydescribed serotype. Polyclonal antisera were generated against arepresentative member of this clade and a comprehensive study ofserologic cross reactivity between the previously described serotypeswas performed. This showed that the new human clade is serologicallydistinct from the other known serotypes and therefore is called Clade F(represented by AAV9).

Rabbit polyclonal antibodies against AAV serotypes 1-9 were generated byintramuscularly inoculating the animals with 1×10¹³ genome copies eachof AAV vectors together with an equal volume of incomplete Freud'sadjuvant. The injections were repeated at day 34 to boost antibodytiters. Serological cross reactivity between AAV 1-9 was determined byassessing the inhibitory effect of rabbit antisera on transduction of293 cells by vectors carrying a reporter gene (AAVCMVEGFP, which carriesenhanced green fluorescent protein) pseudotyped with capsids derivedfrom different AAV sources. Transduction of 84-31 cells by AAVCMVEGFPvectors was assessed under a UV microscope. In assessing serologicrelationships between two AAVs, the ability of both heterologous andhomologous sera to neutralize vectors from each AAV were tested. Ifneutralization by the serum was at least 16-fold lower againstheterologous vectors than homologous vectors in a reciprocal manner, thetwo AAVs are considered distinct serotypes. Neutralization titers weredefined as described previously [(G. P. Gao et al., Proc Natl Acad SciUSA 99, 11854-9 (Sep. 3, 2002)].

TABLE 2 Serologic evaluation of novel AAV vectors Vector pseudotypesused in the neutralization assay from rabbit immunized with: AAV2/1AAV2/2 AAV2/3 AAV2/4 AAV2/5 AAV2/6 AAV2/7 AAV2/8 AAV2/9 AAV2/1 1/163,840No NAB No NAB No NAB 1/40,960 1/40,960 1/40 No NAB No NAB AAV2/2 1/801/81,920 1/5,120 1/20 No NAB 1/80 1/40 1/40 No NAB AAV2/3 1/1,2801/2,560 1/40,960 1/20 1/40 1/2,560 1/1,280 1/1,280 No NAB AAV2/4 1/20 NoNAB No NAB 1/1,280 1/40 No NAB No NAB No NAB 1/40 AAV2/5 1/20,480 No NAB1/80 No NAB 1/163,840 1/5,120 1/40 No NAB No NAB AAV2/6 1/81,920 No NAB1/640 1/40 1/40 1/327,680 1/40 No NAB 1/40 AAV2/7 1/1,280 1/640 1/1,2801/20 No NAB 1/1,280 1/163,840 1/5,120 1/80 AAV2/8 1/20 1/1,280 1/1,280No NAB 1/20 No NAB 1/640 1/327,680 1/2,560 AAV2/9 No NAB No NAB No NABNo NAB No NAB No NAB 1/20 1/640 1/20,480

These data confirm the phylogenetic groupings of the different clonesand clades except for unanticipated serological reactivity of thestructurally distinct AAV5 and AAV1 serotypes (i.e., ratio ofheterologous/homologous titer were 1/4 and 1/8 in reciprocaltitrations).

The result further indicated that AAVhu.14 had a distinct serologicalproperty and did not have significant cross reactivity with antiseragenerated from any known AAV serotypes. The serological distinctivenessof AAVhu.14 was further supported by its uniqueness in the capsidstructure which shared less than 85% amino acid sequence identity withall other AAV serotypes compared in this study. Those findings providedthe basis for us to name AAVhu.14 as a new serotype, AAV9.

EXAMPLE 3 Evaluation of Primate AAVs as Gene Transfer Vectors

The biological tropisms of AAVs were studied by generating vectorpseudotyped in which recombinant AAV2 genomes expressing either GFP orthe secreted reporter gene α-1 antitrypsin (A1AT) were packaged withcapsids derived from various clones and one representative member fromeach primate AAV clade for comparison. For instance, the data obtainedfrom AAV1 was used to represent Clade A, followed by AAV2 for Clade B,Rh.34 for AAV4, AAV7 for Clade D, AAV8 for Clade E, and AAVHu.14 forClade F. AAV5, AAVCh.5 and AAVRh.8 stand as single AAV genotypes for thecomparison.

The vectors were evaluated for transduction efficiency in vitro, basedon GFP transduction, and transduction efficiency in vivo in liver,muscle or lung (FIG. 4).

A. In Vitro

Vectors expressing enhanced green fluorescent protein (EGFP) were usedto examine their in vitro transduction efficiency in 84-31 cells and tostudy their serological properties. For functional analysis, in vitrotransduction of different AAVCMVEGFP vectors was measured in 84-31 cellsthat were seeded in a 96 well plate and infected with pseudotypedAAVCMVEGFP vectors at an MOI of 1×10⁴ GC per cell. AAV vectors werepseudotyped with capsids of AAVs 1, 2, 5, 7, 8 and 6 other novel AAVs(Ch.5, Rh.34, Cy5, rh.20, Rh.8 and AAV9) using the technique describedin G. Gao et al., Proc Natl Acad Sci USA 99, 11854-9 (Sep. 3, 2002).Relative EGFP transduction efficiency was scored as 0, 1, 2 and 3corresponding to 0-10%, 10-30%, 30-70% and 70-100% of green cellsestimated using a UV microscope at 48 hours post infection.

B. In Vivo

For in vivo studies, human α-antitrypsin (A1AT) was selected as asensitive and quantitative reporter gene in the vectors and expressedunder the control of CMV-enhanced chicken β-actin promoter. Employmentof the CB promoter enables high levels of tissue non-specific andconstitutive A1AT gene transfer to be achieved and also permits use ofthe same vector preparation for gene transfer studies in any tissue ofinterest. Four to six week old NCR nude mice were treated with novel AAVvectors (AAVCBhA1AT) at a dose of 1×10¹¹ genome copies per animalthrough intraportal, intratracheal and intramuscular injections forliver, lung and muscle directed gene transfer, respectively. Serumsamples were collected at different time points post gene transfer andA1AT concentrations were determined by an ELISA-based assay and scoredas 0, 1, 2 and 3 relative to different serum A1AT levels at day 28 postgene transfer, depending on the route of vector administration (Liver:0=A1AT<400 ng/ml, 1=A1AT 400-1000 ng/ml, 2=A1AT 1000-10,000 ng/ml,3=A1AT>10,000 ng/ml; Lung: 0=A1AT<200 ng/ml, 1=A1AT 200-1000 ng/ml,2=A1AT 1000-10,000 ng/ml, 3=A1AT>10,000 ng/ml; Muscle: 0=A1AT<100 ng/ml,1=A1AT 100-1000 ng/ml, 2=A1AT 1000-10,000 ng/ml, 3=A1AT>10,000 ng/ml).

A human AAV, clone 28.4/hu.14 (now named AAV9), has the ability totransduce liver at a efficiency similar to AAV8, lung 2 logs better thanAAV5 and muscle superior to AAV1, whereas the performance of two otherhuman clones, 24.5 and 16.12 (hu.12 and hu.13) was marginal in all 3target tissues. Clone N721.8 (AAVrh.43) is also a high performer in allthree tissues.

To further analyze gene transfer efficiency of AAV9 and rh 43 incomparison with that of bench markers for liver (AAV8), lung (AAV5) andmuscle (AAV1), a dose response experiment was carried out. Both newvectors demonstrated at least 10 fold more gene transfer than AAV1 inmuscle, similar performance to AAV8 in liver and 2 logs more efficientthan AAV5 in lung.

A group of AAVs demonstrated efficient gene transfer in all 3 tissuesthat was similar or superior to the performance of their bench marker ineach tissue has emerged. To date, 3 novel AAVs have fallen into thiscategory, two from rhesus (rh10 and 43) and one from human (hu.14 orAAV9). A direct comparison of relative gene transfer efficiency of those3 AAVs to their bench markers in the murine liver, lung and musclesuggests that some primate AAVs with the best fitness might have evolvedfrom rigorous biological selection and evolution as “super” viruses.These are particularly well suited for gene transfer applications.

C. Profiles of Biological Activity

Unique profiles of biological activity, in terms of efficiency of genetransfer, were demonstrated for the different AAVs with substantialconcordance within members of a set of clones or clade. However, invitro transduction did not predict the efficiency of gene transfer invivo. An algorithm for comparing the biological activity between twodifferent AAV pseudotypes was developed based on relative scoring of thelevel of transgene expression and a cumulative analysis of differences.

Cumulative differences of the gene transfer scores in vitro and in vivobetween pairs of AAVs were calculated and presented in the table (ND=notdetermined) according to the following formula. Cumulative functionaldifference in terms of scores between vectors A and B=in vitro(A−B)+lung (A−B)+liver (A−B)+muscle (A−B). The smaller the number, themore similar in function the AAVs. In the grey shaded area, thepercentage difference in sequence is represented in bold italic. Thepercentage difference in cap structure was determined by dividing thenumber of amino-acid differences after a pairwise deletion of gaps by750, the length of the VP1 protein sequence alignment.

These studies point out a number of issues relevant to the study ofparvoviruses in humans. The prevalence of endogenous AAV sequences in awide array of human tissues suggests that natural infections with thisgroup of viruses are quite common. The wide tissue distribution of viralsequences and the frequent detection in liver, spleen and gut indicatethat transmission occurs via the gastrointestinal track and that viremiamay be a feature of the infection.

The tremendous diversity of sequence present in both human and nonhumanprimates has functional correlates in terms of tropism and serology,suggesting it is driven by real biological pressures such as immuneescape. Clearly, recombination contributes to this diversity asevidenced by the second most common human clade, which is a hybrid oftwo previously described AAVs.

Inspection of the topology of the phylogenetic analysis reveals insightinto the relationship between the evolution of the virus and its hostrestriction. The entire genus of dependoviruses appears to be derivedfrom avian AAV consistent with Lukashov and Goudsmit [(V. V. Lukashov,J. Goudsmit, J Virol 75, 2729-40 (March, 2001)]. The AAV4 and AAV5isolates diverged early from the subsequent development of the otherAAVs. The next important node divides the species into two majormonophilic groups. The first group contains clones isolated solely fromhumans and includes Clade B, AAV3 clone, Clade C and Clade A; the onlyexception to the species restriction of this group is the single clonefrom chimpanzees, called ch.5. The other monophilic group, representingthe remaining members of the genus, is derived from both human andnonhuman primates. This group includes Clade D and the rh.8 clone, whichwere isolated exclusively from macaques, and the Clade F, which is humanspecific. The remaining clade within this group (i.e., Clade E) hasmembers from both humans and a number of nonhuman primate speciessuggesting transmission of this clade across species barriers. It isinteresting that the capsid structures of Clade E members isolated fromsome humans are essentially identical to some from nonhuman primates,indicating that very little host adaptation has occurred. Analysis ofthe biology of AAV8 derived vectors demonstrated a broad range of tissuetropism with high levels of gene transfer, which is consistent with amore promiscuous range of infectivity, and may explain its apparentzoonosis. An even greater range and efficiency of gene transfer wasnoted for the Clade F, highlighting the potential for cross speciestransmission, which to date has not been detected.

The presence of latent AAVs widely disseminated throughout human andnonhuman primates and their apparent predisposition to recombine and tocross species barriers raises important issues. This combination ofevents has the potential to lead to the emergence of new infectiousagents with modified virulence. Assessing this potential is confoundedby the fact that the clinical sequalae of AAV infections in primates hasyet to be defined. In addition, the high prevalence of AAV sequences inliver may contribute to dissemination of the virus in the humanpopulation in the setting of allogeneic and xenogenic livertransplantation. Finally, the finding of endogenous AAVs in humans hasimplications in the use of AAV for human gene therapy. The fact thatwild type AAV is so prevalent in primates without ever being associatedwith a malignancy suggests it is not particularly oncogenic. In fact,expression of AAV rep genes has been shown to suppress transformation P.L. Hermonat, Virology 172, 253-61 (September, 1989)].

EXAMPLE 4 AAV 2/9 Vector for the Treatment of Cystic Fibrosis AirwayDisease

To date, CFTR gene transfer to the lung for the treatment of CF airwaydisease has been limited by poor vector performance combined with thesignificant barriers that the airway epithelium poses to effective genetransfer. The AAV2 genome packaged in the AAV9 capsid (AAV2/9) wascompared to AAV2/5 in various airway model systems.

A 50 μl single dose of 1×10¹¹ genome copies (gc) of AAV2/9 expressingeither the nuclear targeted β-galactosidase (nLacZ) gene or the greenfluorescence protein (GFP) gene under the transcriptional control of thechicken β-actin promoter was instilled intranasally into nude and alsoC57Bl/6 mice. Twenty-one days later, the lung and nose were processedfor gene expression. In control animals transduced with AAV2/9-GFP, noLacZ positive cells were seen. AAV2/9-nLacZ successfully transducedmainly airways, whereas AAV2/5-nLacZ transduced mainly alveoli and fewairways. Across the nasal airway epithelium, both AAV2/5 and AAV2/9transduced ciliated and non-ciliated epithelial cells.

Epithelial cell specific promoters are currently being evaluated toimprove targeting to the airway cells in vivo. Based on the in vivofindings, the gene transfer efficiency of AAV2/9 to human airwayepithelial cells was tested next. Airway epithelial cells were isolatedfrom human trachea and bronchi and grown at air-liquid-interface (ALI)on collagen coated membrane supports. Once the cells polarized anddifferentiated, they were transduced with AAV2/9 or AAV2/5 expressingGFP from the apical as well as the basolateral side. Both AAV2/5 andAAV2/9 were successful at transducing epithelial cells from thebasolateral surface. However, when applied onto the apical surfaceAAV2/9 resulted in a 10-fold increase in the number of transduced cellscompared to AAV2/5. Currently, the gene transfer performance of AAV2/9in the lungs and nasal airways of nonhuman primates is being evaluated.

This experiment demonstrates that AAV2/9 can efficiently transduce theairways of murine lung and well-differentiated human airway epithelialcells grown at ALI.

EXAMPLE 5 Comparison of Direct Injection of AAV1(2/1) and AAV9(2/9) inAdult Rat Hearts

Two adult (3 month old) rats received a single injection of 5×10¹¹particles of AAV2/1 or AAV2/9 in the left ventricle

The results were spectacular, with significantly more expressionobserved in the adult rat heart with AAV2/9 vectors as compared toAAV2/1, as assessed by lacZ histochemistry. AAV2/9 also shows superiorgene transfer in neonatal mouse heart.

EXAMPLE 6 AAV2/9 Vector for Hemophilia B Gene Therapy

In this study, AAV 2/9 vectors are shown to be more efficient and lessimmunogenic vectors for both liver and muscle-directed gene therapy forhemophilia B than the traditional AAV sources.

For a liver-directed approach, evaluation of the AAV2/9 pseudotypedvector was performed in mouse and dog hemophilic models. Inimmunocompetent hemophilia B mice (in C57BL/6 background), long-termsuperphysiological levels of canine Factor IX (cFIX, 41-70 μg/ml) andshortened activated partial thromboplastin time (aPTT) have beenachieved following intraportal injection of 1×10¹¹ genome copies(GC)/mouse of AAV2/7, 2/8, and 2/9 vectors in which the cFIX isexpressed under a liver specific promoter (LSP) and woodchuck hepatitisB post-transcriptional responsive element (WPRE). A 10-fold lower dose(1×10¹⁰ GC/mouse) of AAV2/8 vector generated normal level of cFIX andaPTT time. In University of North Caroline (UNC) hemophilia B dogs, itwas previously demonstrated that administration of an AAV2/8 vector intoa dog previously treated with an AAV2 vector was successful; cFIXexpression peaked at 10 μg/ml day 6 after the 2^(nd) intraportalinjection (dose=5×10¹² GC/kg), then gradually decreased and stabilizedaround 700 ng/ml (16% of the normal level) throughout the study (1½years). This level was about 3-fold higher than that from a hemophilia Bdog that received a single injection of AAV2-cFIX at the similar dose.Recently, two naïve hemophilia B dogs were injected with AAV2/8 vectorsintraportally at the dose of 5.25×10¹² GC/kg. cFIX levels in one dog(male) reached 30% of normal level (1.5 μg/ml) ten weeks after injectionand has sustained at 1.3-1.5 μg/ml, while the second dog (female)maintained cFIX expression at about 10% of normal level. Whole bloodclotting time (WBCT) and aPTT were both shortened after the injection,suggesting the antigen was biologically active. Liver enzymes (aspartateamino transferase (SGOT), alanine amino transferase (SGPT) in both dogsremained in the normal range after surgery. These AAV were alsoevaluated for muscle-targeted gene therapy of hemophilia B.AAV-CMV-cFIX-WPRE [an AAV carrying cFIX under the control of a CMVpromoter and containing the WPRE] packaged with six different AAVsources were compared in immunocompetent hemophilia B mice (in C57BL/6background) after intramuscular injection at the dose of 1×10¹¹GC/mouse. cFIX gene expression and antibody formation were monitored.Highest expression was detected in the plasma of the mice injected withAAV2/8 vectors (1460±392 ng/ml at day 42), followed by AAV2/9 (773±171ng/ml at day 42) and AAV2/7 (500±311 ng/ml at day 42). Levels weremaintained for 5 months. Surprisingly, cFIX expression by AAV2/1 rangedfrom 0-253 ng/ml (average: 66±82 ng/ml). Anti-cFIX inhibitor (IgG) wasdetected in some of the AAV2/1-injected mice. cFIX expression levels inthese mice correlated well with inhibitor levels. Further screening ofinhibitor formation was performed on day 28 samples for all AAV.Hemophilia B mice showed highest inhibitor formation against AAV2/2,followed by AAV2/5, and AAV2/1. Only sporadic and low level inhibitorswere detected in animals injected with AAV2/7, AAV2/8 and AAV2/9. Thus,the advantages of the new AAV serotype 2/9 vectors for muscle-directedgene therapy for hemophilia B as more efficient and safe vectors withouteliciting any significant anti-FIX antibody formation are shown.

EXAMPLE 7 Novel Rh.43 Vectors of Invention

A. Comparison of AAVrh.43 Based A1AT Expression Vector with AAV8 andAAV9 in Mouse Liver Directed Gene Transfer

Novel AAVrh.43, which belongs to Clade E by phylogenetic analysis vectorwas compared to AAV8 and novel AAV9 for hA1AT levels after intraportalinfusion to the mouse liver. More particularly, pseudotyped AAVrh.43,AAV2/8 and AAV2/9 vectors were compared in mouse liver-directed genetransfer. Pseudotyped vectors at doses of 1×10¹¹ GC, 3×10¹⁰ GC and1×10¹⁰ GC per animal were administrated to 4-6 week old C57BL/6 mouseintramuscularly. Serum samples were collected from animals at day 28post vector infusion for the human alpha 1 anti-trypsin (hA1AT) assay.

The data indicated that the novel AAVrh.43 vector had indeed aperformance similar to that of AAV9 in the mouse model.

B. Nuclear Target LacZ Gene Transfer to Mouse Liver and Muscle Mediatedby Pseudotyped AAV Vectors.

Novel AAV9 and AAVrh.43 based vectors of the invention were compared toAAV1 and AAV2-based vector. The vectors were injected at a dose of1×10¹¹ GC per mouse either intraportally to target liver orintramuscularly to the right anterior tibialis muscle of C57BL/6 miceintramuscularly. The animals were sacrificed at day 28 post genetransfer and tissues of interest harvested for X-gal histochemicalstaining.

The AAVrh.43 vector demonstrated gene transfer efficiency that was closeto AAV9 but at least 5 fold higher than AAV1. The property of AAVrh.43was further analyzed in both liver and muscle using nuclear targetedLacZ gene as a reporter to visualize extend of gene transferhistochemically.

C. Comparison of AAVrh.43 Based A1AT Expression Vector with AAV5 inMouse Lung Directed Gene Transfer

A novel rh.43-based vector of the invention also demonstrated superbgene transfer potency in lung tissue. Different doses (1×10¹⁰, 3×10¹⁰and 1×10¹¹ GC per animal) of pseudotyped vectors were administrated to4-6 week old C57BL/6 mouse lungs intratracheally. Serum samples werecollected from animals at different time points for hA1AT assay.

This vector was compared to AAV5 at different doses for levels of hA1ATdetected systematically after intratracheal installation to the mouselung. The data indicated that this novel vector was at lease 100 foldmore efficient than AAV5 in the mouse model.

EXAMPLE 8 Novel Human AAV Based Vectors in Mouse Models for Liver andLung-Directed Gene Transfer

The human clones, AAVhu.37, AAVhu.41 and AAVhu.47 were pseudotyped andexamined for gene transfer potency in mouse tissues. AAVCBA1AT vectorspseudotyped with capsids of hu.37, hu.41 and hu.47 were prepared usingthe methods described herein and administrated to 4-6 week old C57BL/6mouse through intraportal and intratracheal injections. Serum sampleswere collected from animals at day 14 post vector injection for hA1ATassay, which was performed in accordance with published techniques.AAVhu.47 belongs to AAV2 family (clade B) AAV2 and was isolated from ahuman bone marrow sample. AAVhu.37 and AAVhu.41 came from a human testistissue and a human bone marrow sample respectively. Phylogenetically,they fall into the AAV 8 clade (clade E).

Serum A1AT analysis of injected animals indicated that AAV hu.41 and AAVhu.47 performed poorly in the three tissues tested. However, genetransfer potency of AAVhu.37 derived vector was similar to that of AAV8in liver and AAV9 in lung

All publications cited in this specification are incorporated herein byreference. While the invention has been described with reference toparticularly preferred embodiments, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention.

1. An AAV comprising an AAV9 capsid, wherein the AAV9 capsid comprisesan amino acid sequence having at least 95% identity with amino acids 203to 736 of SEQ ID NO: 123, and wherein said AAV further comprises aminigene having AAV inverted terminal repeats and a transgene comprisinga heterologous gene operably linked to regulatory sequences which directexpression of the heterologous gene in an host cell.
 2. The AAVaccording to claim 1, wherein the AAV9 capsid comprises a sequenceselected from the group consisting of an amino acid sequence having atleast 95% identity with amino acids 138-736 of SEQ ID NO: 123 and anamino acid sequence having at least 95% identity with amino acids 1-736of SEQ ID NO:
 123. 3. The AAV according to claim 1, wherein the AAV9capsid comprises a sequence selected from the group consisting of anamino acid sequence having at least 99% identity with amino acids203-736, an amino acid sequence having at least 99% identity with aminoacids 138-736, and an amino acid sequence having at least 99% identitywith amino acids 1-736.
 4. A composition comprising an AAV vectoraccording to claim 1 and a physiologically compatible carrier.
 5. Anadeno-associated virus (AAV) comprising an AAV capsid selected from thegroup consisting of: serotype hu.35, amino acids 204 to 734 of SEQ IDNO: 186; serotype hu.37, amino acids 204 to 738 of SEQ ID NO: 88;serotype pi.2, amino acids 203 to 731 of SEQ ID NO: 95; serotype rh.64,SEQ ID NO: 99; and serotype rh.65R2, SEQ ID NO: 233; and wherein the AAVfurther comprises a minigene having AAV inverted terminal repeats and aheterologous gene operably linked to regulatory sequences which directits expression in a host cell.
 6. An adeno-associated virus (AAV) cladeF comprising AAV9, AAV hu.14/AAV9 (SEQ ID Nos: 3 and 123), hu.31 (SEQ IDNOs: 1 and 121) and hu.32 (SEQ ID Nos: 2 and 122), wherein each memberof said AAV clade is phylogenetically related as determined using aNeighbor-Joining heuristic by a bootstrap value of at least 75% per 1000isolates and a Poisson correction distance measurement of no more than0.05.
 7. The AAV clade F according to claim 6 wherein said cladecomprises at least AAV hu.14/AAV9 (SEQ ID Nos: 3 and 123), hu.31 (SEQ IDNOs: 1 and 121) and hu.32 (SEQ ID Nos: 2 and 122).
 8. A compositioncomprising a pharmaceutical carrier and a protein comprising anAAV9/HU.14 capsid protein or a fragment thereof selected from the groupconsisting of: vp1 capsid protein, amino acids (aa) 1 to 736, SEQ IDNO:123; an AAV capsid comprising a vp2 capsid protein, aa 138 to 736,SEQ ID NO: 123; an AAV capsid comprising a vp3 capsid protein, aa 203 to736, SEQ ID NO: 123; an AAV capsid comprising a fragment encompassing atleast one hypervariable region (HVR)1 through 12 selected the groupconsisting of: aa 146 to 152; aa 182 to 187; aa 262 to 264; aa 263 to266; aa 263 to 266; aa 381 to 383; 383 to 385; aa 450 to 474; aa 451 to475; aa 490 to 495; aa 491 to 496; aa500 to 504; aa 501 to 505; aa 514to 522; aa 533 to 554; aa 534 to 555; aa 581 to 594; aa 583 to 596; aa658 to 667; aa 660 to 669; and aa 705 to 719; aa 707 to 723; aa 24 to42, aa 25 to 28; aa 81 to 85; aa133 to 165; aa 134 to 165; aa 137 to143; aa 154 to 156; aa 194 to 208; aa 261 to 274; aa 262 to 274; aa 171to 173; aa 185 to 198; aa 413 to 417; aa 449 to 478; aa 494 to 525; aa534 to 571; aa 581 to 601; aa 660 to 671; aa 709 to 723; and aa 1 to184, aa 199 to 259; aa 274 to 446; aa 603 to 659; aa 670 to 706; aa 724to 736; aa 185 to 198; aa 260 to 273; aa447 to 477; aa495 to 602; aa603to 659; aa 660 to 669; and aa707 to 723, wherein the amino acid numbersare those of the AAV2 capsid, SEQ ID NO:4 and corresponding regions inthe capsid of AAV9/HU.14, SEQ ID NO:123.
 9. A vector containing anucleic acid sequence encoding a protein according to claim
 8. 10. Thevector according to claim 9, wherein said nucleic acid sequence isselected from the group consisting of: the nucleotides (nt) 1 to 2211 ofSEQ ID NO: 3; the nt 411 to 2211 of SEQ ID NO: 3; and the nt 609 to 2211of SEQ ID NO:
 3. 11. The vector according to claim 10, wherein saidmolecule comprises an AAV sequence encoding an AAV capsid protein and afunctional AAV rep protein.
 12. The vector according to claim 10,wherein said molecule is a plasmid.
 13. A method of generating arecombinant adeno-associated virus (AAV) comprising an AAV capsidcomprising the steps of culturing a host cell containing: (a) a moleculeencoding an AAV capsid protein; (b) a functional rep gene; (c) aminigene comprising AAV inverted terminal repeats (ITRs) and atransgene; and (d) sufficient helper functions to permit packaging ofthe minigene into the AAV capsid protein, wherein said host cellcomprises a nucleic acid sequence according to claim
 10. 14. A host celltransfected with an adeno-associated virus according to claim
 1. 15. Ahost cell transfected with the vector according to claim 9.